DE69504808T2 - CORTICOTROPIN RELEASING RECEPTORS OF FACTOR 2 - Google Patents

Abstract

The present invention provides isolated nucleic acid molecules encoding CRF<SUB>2 </SUB>receptors, recombinant expression vectors and host cells suitable for expressing such receptors, as well as compositions and methods which utilize such receptors.

Description

Technical area

The present invention relates generally to cell surface receptors and specifically to corticotropin releasing factor2 receptors.

Background of the invention

Corticotropin-releasing factor ("CRF") is a 41-amino acid peptide that was originally isolated from the hypothalamus due to its ability to stimulate the production of adrenocorticotropic hormone ("ACTH") and other proopiomelanocortin ("POMC") products of the anterior pituitary (Vale et al., Science 213: 1394-1397, 1981). Briefly, CRF is believed to initiate its biological effects by binding to a plasma membrane receptor that is ubiquitously distributed in the brain (DeSouza et al., Science 224:1449-1451, 1984), the pituitary gland (Wynn et al., Biochem. Biophys. Res. Comm. 110:602-608, 1983), the adrenal glands (Udelsmann et al., Nature 319:147-150, 1986), and the spleen (Webster, E.L., and E.B. DeSouza, Endrocrinology 122:609-617, 1988). This receptor is bound to a GTP-binding protein (Pernn et al., Endocrinology 118:1171-1179, 1986) that mediates the CRF-stimulated increase in intracellular cAMP (Bilezikjian, L.M. and W.W. Vale, Endocrinology 113:657-662, 1983).

In addition to its role in stimulating the production of ACTH and POMC, CRF is also thought to coordinate many of the endocrine, autonomic, and behavioral responses to stress and may be involved in the pathophysiology of affective disorders. In addition, CRF is believed to be a key intermediate in the communication between the immune, central nervous, endocrine, and cardiovascular systems (Crofford et al., J. Clin. Invest. 90:2555-2564, 1992; Sapolsky et al., Science 238:522-524, 1987, Tilders et al., Regul. Peptides 5:77-84, 1982; Fisher et al., Reg. Peptide 5:153-161, 1983).

A receptor for CRF has been cloned from rat (Pernn et al., Endo 133(6):3058-3061, 1993) and human brain (Chen et al., PNAS 90(19):8967-8971, 1993; Vita et al., FEBS 335(1):1-5, 1993). This receptor is a 415 amino acid protein comprising seven membrane-spanning domains and has a predicted molecular weight of 44,000 daltons. Identity comparison between rat and human sequences shows a high degree of homology (97%) at the amino acid level. In addition, Scatchard analysis of recombinantly produced human receptor reveals a single component site with a high affinity for CRF (Kd of 1.6 ± 0.3 nM).

The present invention provides new, previously unidentified CRF receptors, termed "corticotropin releasing factor₂ ("CRF₂"), corticotropin releasing factor receptors. In addition, the present invention provides compositions and methods using such CRF₂ receptors, as well as other related advantages.

Summary of the invention

According to the present invention there is provided an isolated nucleic acid molecule encoding a CRF₂ receptor, wherein the CRF₂ receptor is encoded by:

(a) a nucleic acid sequence with the coding region of one of the sequences ID Nos. 1, 3 or 7 or a derivative thereof with a homology with the coding region of greater than 70%

(b) a nucleic acid sequence capable of hybridising under high stringency conditions to a nucleic acid sequence complementary to (a); or

(c) nucleic acid sequences which are degenerate as a result of the genetic code for CRF2 receptors encoded by a nucleic acid sequence as defined in (a) or (b).

According to the present invention there is further provided an isolated nucleic acid molecule encoding a CRF₂ N-terminal extracellular domain, wherein the CRF₂ N-terminal extracellular domain is encoded by:

(a) a nucleic acid sequence derived from the region encoding the N-terminal extracellular domain defined by nucleotide No. 44 to nucleotide No. 457 of Sequence I.D. No. 1, nucleotide No. 216 to nucleotide No. 569 of Sequence I.D. No. 3, or the equivalent region of Sequence I.D. No. 7, or a derivative thereof having more than 70% homology with the coding region;

(b) a nucleic acid sequence capable of hybridising under conditions of high stringency to a nucleic acid sequence complementary to (a); or

(c) nucleic acid sequences that are degenerate as a result of the genetic code for CRF2 receptors encoded by a nucleic acid sequence as defined in (a) or (b).

Briefly, the present invention provides compositions and methods that utilize CRF₂ receptors (also referred to as "CRF₂-corticotropin releasing factor receptors" or "CRF₂R"). Within one aspect of the present invention, isolated nucleic acid sequences encoding CRF₂ receptors are provided. In one embodiment, nucleic acid molecules encoding a CRF₂ receptor are provided, such as that disclosed in Sequence I.D. No. 4, from amino acid 1 to amino acid 411. In one embodiment, nucleic acid molecules comprising the nucleotide sequence in Sequence I.D. No. 3 from nucleotide No. 216 to nucleotide No. 1448 are provided. In another embodiment, nucleic acid molecules are provided that encode a CRF2 receptor, such as that disclosed in Sequence I.D. No. 2, from amino acid 1 to amino acid 431. In another embodiment, nucleic acid molecules are provided that comprise the nucleotide sequence in Sequence I.D. No. 1, from nucleotide number 44 to nucleotide number 1336. Nucleic acid sequences encoding CRF2 receptors of the present invention can be isolated from virtually any warm-blooded animal, including, for example; humans, macaques, horses, cattle, sheep, pigs, dogs, cats, rats, and mice.

In a further aspect of the present invention, isolated nucleic acid molecules are provided which encode parts of a CRF₂ receptor, such as the N-terminal extracellular domain. In one embodiment, isolated nucleic acid molecules are provided which comprise the nucleotide sequence in Sequence ID No. 3 from nucleotide No. 216 to nucleotide No. 569. In another embodiment, isolated nucleic acid molecules are provided which encode a protein with the amino acid sequence of Sequence ID No. 4 from amino acid No. 1 to amino acid No. 118. In a further embodiment, isolated nucleic acid molecules are provided which comprise the nucleotide sequence in Sequence ID No. 1 from nucleotide No. 44 to nucleotide No. 457. In a further embodiment, isolated nucleic acid molecules are provided which encode a protein with the amino acid sequence of Sequence ID No. 2 from amino acid No. 1 to amino acid No. 138.

In another embodiment of the invention, expression vectors are provided that are capable of expressing the nucleic acid molecules described above. In further aspects, recombinant viral vectors are provided that are capable of directing the expression of the nucleic acid molecules described above. Typical examples of such viral vectors include retroviral vectors, adenoviral vectors, and herpes simplex virus vectors. The present invention also provides host cells containing the expression vectors described above, as well as the receptor or portions thereof encoded by the nucleic acid molecules described above. In other embodiments, isolated portions of CRF2 receptors are provided, including, for example, isolated portions of extracellular domains, such as the N-terminal extracellular domain.

In further aspects of the invention, isolated antibodies are provided that are capable of specifically binding to the CRF2 receptors described above. In one embodiment, the antibodies can be selected from the group comprising polyclonal antibodies, monoclonal antibodies and antibody fragments. In other embodiments, antibodies are provided that are capable of blocking the binding of CRF (or other substrates such as sauvagin or urotensin I) to a CRF2 receptor. In preferred embodiments, the antibodies can be selected from the group comprising murine and human antibodies. In preferred embodiments of the invention, the antibodies specified above are produced by hybridomas.

In yet another aspect of the present invention, nucleic acid molecules are provided which are capable of specifically binding to nucleic acid molecules encoding any of the CRF₂ receptors described above. Such molecules may be between at least "y" nucleotides in length, where "y" is an integer between 14 and 1230, and, further, suitably selected for use as probes or primers as described below. Particularly preferred probes of the present invention are at least 18 nucleotides in length.

In further aspects of the present invention, methods are provided for detecting the presence of a compound that binds to a CRF2 receptor comprising the steps of (a) exposing one or more compounds to cells that exhibit CRF2 receptors under conditions and for a time sufficient to allow binding of the compounds to the receptors and (b) isolating compounds that bind to the receptors so that the presence of a compound that binds to a CRF2 receptor can be detected. In another aspect, methods are provided for detecting the presence of a compound that binds to a CRF2 receptor comprising the steps of (a) exposing one or more compounds to an N-terminal CRF2 receptor extracellular domain under conditions and for a time sufficient to permit binding of a compound to the N-terminal extracellular domain and (b) isolating compounds that bind to the N-terminal CRF2 receptor extracellular domain so that the presence of a compound that binds to a CRF2 receptor can be detected. In one embodiment, the compounds are labeled with an agent selected from the group comprising fluorescent molecules, enzymes, and radionuclides.

In another aspect of the present invention, methods are provided for determining whether a selected compound is a CRF2 receptor agonist or antagonist comprising the steps of (a) exposing a selected compound to cells expressing CRF2 receptors under conditions and for a time sufficient to allow binding of the compound and an associated response in intracellular titers of cAMP, and (b) detecting either an increase or decrease in the titer of intracellular cAMP, thereby determining whether the selected compound is a CRF2 receptor agonist or antagonist.

Within further aspects, methods are provided for detecting the presence of a CRF₂ receptor agonist or antagonist in a pool of compounds comprising the steps of (a) exposing a pool of compounds to cells expressing CRF₂ receptors under conditions and for a time sufficient to induce binding of the compound and an associated response in intracellular titers of cAMP and (b) isolating compounds which either increase or decrease the intracellular titer of cAMP so that the presence of a CRF₂ receptor agonist or antagonist can be detected.

In another aspect, methods are provided for determining whether a selected compound is a CRF2 receptor antagonist comprising the steps of (a) exposing a selected compound in the presence of a CRF2 receptor agonist to a recombinant CRF2 receptor coupled to a pathway under conditions and for a time sufficient to permit binding of the compound to a receptor and an associated response by the pathway, and (b) detecting a reduction in stimulation of the pathway resulting from binding of the compound to the CRF2 receptor relative to stimulation of the pathway by the CRF2 receptor agonist alone, thereby determining the presence of a CRF2 antagonist. In other aspects, methods are provided for determining whether a selected compound is a CRF2 receptor agonist comprising the steps of (a) exposing a selected compound to a CRF2 receptor coupled to a pathway under conditions and for a time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, and (b) detecting an increase in stimulation of the pathway, compared to the activity of the pathway prior to step (a), resulting from binding of the compound to the CRF2 receptor and therefrom determining the presence of a CRF2 receptor agonist.

In other aspects of the present invention, methods are disclosed for treating CRF2 receptor-associated disorders where it is desired to either increase or decrease stimulation of a CRF2 receptor response pathway. For example, in one aspect, methods are provided for treating cerebrovascular disorders such as ictus, reperfusion injury, and migraines comprising the steps of: administering to a patient a therapeutically effective amount of a CRF2 receptor antagonist such that the disorder is cured or alleviated. In other methods, methods are disclosed for treating learning or memory disorders comprising administering to a patient a therapeutically effective amount of CRF2 receptor antagonists. In still other aspects, methods are disclosed for treating Alzheimer's disease comprising administering to a patient a therapeutically effective amount of a CRF2 receptor antagonist. receptor antagonists to a patient. Typical examples of suitable CRF₂ receptor antagonists include α-helical oCRF (9-41) or d-Phe r/h CRF (12- 41).

These and other aspects of the present invention will become apparent upon reference to the following detailed description and the accompanying drawings. In addition, various references are provided below which describe in detail certain procedures or compositions (e.g., plasmids, etc.) and are therefore incorporated by reference in their entirety.

Short description of the drawings

Figure 1 schematically illustrates the structure of an exemplary CRF2 receptor (CRF2β; Sequence ID No. 2).

Figure 2 schematically illustrates the structure of a second exemplary CRF2 receptor (CRF2α; Sequence ID No. 4).

Figure 3 is a graph depicting cAMP accumulation in cells transfected with CRF1 receptor.

Figure 4 is a graph depicting cAMP accumulation in cells transfected with CRF2 receptor.

Figure 5 is a graph depicting the effect of the antagonists α-helical and d-Phe on sauvagine -stimulated cAMP production in CRF1 -transfected cells.

Figure 6 is a graph depicting the effect of the antagonists α-helical and d-Phe on sauvagine -stimulated cAMP production in CRF2α-transfected cells.

Figure 7 is a photograph of an RNase protection assay of two CRF2 subtypes (CRF2α and CRF2β) and β-actin. Ts = testis; Ht = heart; Sk = skeletal muscle; Lv = liver; Kd = kidney; Sp = spleen; Ol = olfactory bulb; St = striatum; Hy = hypothalamus; Pt = pituitary gland; Cx = cortex; Hp = hippocampus.

Figure 8A, A', B, B', C, and C' are a series of photographs showing the anatomical distribution of two CRF2 subtypes. Figure 8A, B, and C are coronal sections of rat brain probed with CRF2α and CRF2ß. Figure 8A', B', and C' are adjacent sections probed with CRF2ß antisense cRNA.

Figure 9 is a schematic representation of the nucleotide sequence homology between CRF1 and CRF2 receptors across the coding region of the receptors. Lower bars indicate the region of the receptors against which CRF1 and CRF2 cRNA probes were designed.

Figure 10 shows two-color-coded digitized images of CRF1 and CRF2 receptor mRNA expression in adjacent horizontal brain sections. Regions showing high levels of mRNA expression are coded in red and orange, whereas the lowest levels of expression are coded in blue.

Fig. 11A, B, C, D, and E are a series of photographs showing the rostrocaudal (A-E) distribution of CRF2 receptor mRNA (left hemisphere) and CRF1 receptor mRNA (right hemisphere) in digitized coronal brain sections. Epy, ependymal layer of olfactory bulb; Int Gr, inner granule cell layer of olfactory bulb; G1, granule cell layer of olfactory bulb; LSI, lateral septal nucleus (intermediate part); LSV, lateral septal nucleus (ventral part); MS, medial septal nucleus; Fr Ctx, stim cortex; Pir, piriform cortex; CA1, field CA1 (Ammonshom); CA3, field CA3 (Ammon’s horn); DG, dentate gyrus; Chp, choroid plexus; MeA, medial amygdaloid nucleus; VMH, ventromedial hypothalamic nucleus; Cing Ctx, cingulate cortex; BLA, basolateral amygdaloid nucleus; Pco, posterior cortical amygdaloid nucleus; RN, red nucleus; Oc Ctx, occipital cortex; MG, medial geniculate nucleus; PDTg, posterior dorsal tegmental nucleus; Trg Nuc, trigeminal nucleus; Pn, pontine gray.

Fig. 12A and B are two darkfield photomicrographs of cells hybridized with (35S)-cRNA-CRF2 probe in (A) the intermediate (LSI) and ventral (LSV) lateral septal nucleus. At high resolution (B), note the high level of CRF2 receptor expression in cells in the ventral lateral septum. Cau, caudate; LV, lateral ventricle.

Figure 13A, B and C are a series of darkfield photomicrographs of cells hybridized with (A) (35S)-cRNA-CRF probe, (B) (35S)-cRNA-CRF2 probe and (C) (35S)-cRNA-CRF1 probe in adjacent coronal sections through the bed nucleus of the stria terminalis (BNST). Note in (A) the higher concentration of CRF-expressing cells in the posterolateral area (BNSTp1), whereas in (B) CRF2 receptor expression is predominantly localized to the medial aspects of the nucleus (BNSTpm). In (C), CRF1 receptor mRNA expression is evident in both medial and lateral layers of the nucleus. Fx, Formx.

Fig. 14A and B are two darkfield photomicrographs of cells hybridized with (A) (35S)-cRNA-CRF2 probe and (B) (35S)-cRNA CRF1 probe in the anterior cortical amygdaloid nucleus (ACo). Note in (A) the high level of CRF2 receptor mRNA expression in cells throughout the nucleus, whereas in (B) CRF1 receptor expression is comparable to background signal. CRF2 receptor mRNA expression is also evident within the supraoptic nucleus in (A), an area where CRF1 receptor expression is undetectable (B). opt, optic pathway, arrows in (A) indicate the cut edge.

Fig. 15A, B, and C are a series of darkfield photomicrographs of cells hybridized with (A) (35S)-cRNA-CRF1 probe and (B) and (C) (35S)-cRNA-CRF2 probe in the hippocampal formation. Within the dosal hippocampus, both CRF1 and CRF2 receptor expression was comparatively low. However, within the ventral hippocampus (C), cells showing high levels of CRF2 receptor mRNA expression were evident in the dentate gyrus and subiculum. *, emulsion artifact.

Fig. 16A and B are two darkfield photomicrographs of cells hybridized with (35S)-cRNA-CRF2 probe in the ventromedial hypothalamic nucleus (VMH) (A) and (B). Note at high resolution (B) the high level of CRF2 receptor expression in both the dorsomedial (DM) and ventrolateral (VL) aspects of the nucleus. 3v, third ventricle; opt, optic pathway.

Fig. 17A, B, C and D are a series of darkfield photomicrographs of cells probed with (35S)-cRNA-CRF2 probe (A) and (C) and (35S)-cRNA-CRF1 probe (B) and (D) in adjacent coronal sections through the supraoptic nucleus (SO) and suprachiasma tic nucleus (Sch). Note the absence of CRF₁ receptor expression in either nucleus (B) or (D). Opt, optic pathway; *, labeled arteriole.

Fig. 18A, B, and C are a series of darkfield photomicrographs of cells hybridized with (A) (35S)-cRNA-CRF probe, (B) (35S)-cRNA-CRF2 probe, and (C) (35S)-cRNA-CRF1 probe in adjacent sections through the paraventricular nucleus of the hypothalamus. In (A), CRF-expressing cells are evident within the medial and dorsal aspects of the paraventricular nucleus. Note in (B) that CRF2 receptor expression is strongest in the medial parvocellular area (mpv) with only scattered labeled cells evident in the dorsal subdivision. CRF1 - Receptor expression is not noticeable in either subdivision (C). 3v, third ventricle; Fx, Formx.

Fig. 19A, B, and C are a series of darkfield photomicrographs of cells hybridized with (35S)-cRNA-CRF2 probe in (A) dorsal raphe (DR) and central gray (CG), (B) median raphe (MnR), and (C) interpeduncular nucleus (IPN).

Figure 20A is a photomicrograph showing CRF2 receptor mRNA expression in the choroid plexus (ChP). Figure 20B is a photomicrograph showing an adjacent Nissl stained section. 4v, fourth ventricle.

Figure 21A is a high-magnification darkfield image of CRF2 receptor mRNA expression in a cerebral arteriole. Figure 21B is a brightfield Nissel-stained section of the arteriole shown in (A). Note the characteristic muscular arteriolar wall in (B).

Fig. 22A, B, C, and D are a series of darkfield photomicrographs of CRF1 receptor mRNA expression (A) and (B) and CRF2 receptor mRNA expression (C) and (D) in the anterior lobe (AL) of the pituitary gland. Note in (A) the clustering of cells expressing CRF1 mRNA, presumably reflecting the distribution of pituitary corticotropes, whereas CRF2 mRNA expression is present only in scattered cells (C). At high resolution, a prominent accumulation of silver grains is evident over anterior lobe cells hybridized with (35S)-cRNA-CRF1 probe, arrow in (B), whereas only faint accumulations of silver grains were evident over cells hybridized with (35S)-cRNA-CRF2 probe, arrow in (D).

Detailed description of the invention Definitions

Before setting forth the invention, it may be helpful to understand it to provide the definitions of certain terms used herein.

"CRF2 receptors" (also called "CRF2 corticotropin releasing factor receptors" or "CRF2R"), as used herein, refers to receptor proteins that bind corticotropin releasing factor and other proteins such as urotensin I and sauvagin. CRF2 receptors can be distinguished from other receptors, such as corticotropin releasing factor receptors, based on criteria such as substrate binding affinity, tissue distribution, and sequence homology. For example, CRF2 receptors of the present invention are more than 70% homologous, preferably more than 75-80% homologous, more preferably more than 85-90% homologous, and most preferably more than 92%, 95%, or 97% homologous to the CRF2 receptors disclosed herein (e.g., Sequence I.D. No. 3). CRF2 receptors in their native configuration are believed to exist as membrane-bound proteins consisting of an N-terminal extracellular domain, seven transmembrane domains separated by three intracellular and three extracellular loops, and a C-terminal intracellular domain (see Figures 1 and 2). As used in the context of the present invention, CRF2 receptors should be understood to include not only the proteins disclosed herein (see Sequence Idea Nos. 2, 4 and 8), but also substantially similar derivatives and analogs as discussed below.

"Nucleic acid molecule" means a nucleic acid sequence in the form of a separate fragment or as a component of a larger nucleic acid construct derived from the nucleic acid which has been isolated at least once in substantially pure form, i.e., free from contaminating endogenous materials, and in an amount or concentration which allows identification, manipulation and recovery of the sequence and its component nucleotide sequences by standard biochemical techniques, e.g., using a cloning vector. Such sequences are preferably provided in the form of an open reading frame which is not interrupted by internal untranslated sequences, or introns, which are typical in eukaryotic genes. Genomic nucleic acid containing the relevant sequences may also be used. Sequences of untranslated nucleic acid may be present 5' or 3' of the open reading frame where the same will not interfere with manipulation or expression of the coding regions.

"Recombinant expression vector" means a replicable nucleic acid construct that is used to either amplify or express nucleic acid sequences encoding CRF2 receptors. This construct comprises an assembly of (1) a genetic element or elements having a regulatory function in gene expression, e.g., promoters, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences.

As noted above, the present invention provides isolated nucleic acid molecules encoding CRF2 receptors. Briefly, CRF2 receptors are G-coupled protein receptors capable of binding a substrate (such as CRF, or other substrates such as sauvagine and urotensin I) and transducing the signal delivered to the cell by the substrate. Such signal transduction typically occurs when a response pathway is activated by an external stimulus that is generally, but not always, directly coupled to a membrane-bound receptor. Response pathways generally induce cellular responses such as secretion of extracellular matrix from responding cell lines, hormone secretion, chemotaxis, differentiation, or the initiation or inhibition of cell division by responding cells. As used herein, coupling of receptors to pathways refers to the direct activation of a pathway or the transduction of a signal via a second messenger, such as a G protein, to activate a cellular pathway.

A typical CRF₂ receptor that can be obtained using the methods described herein (see Example 1) is schematically shown in Fig. 1 (see also Fig. 2). Briefly, this CRF₂ receptor is composed of an extracellular N-terminal domain (amino acids 1-117), a first transmembrane domain (amino acids 118-138), a first intracellular domain (139-147), a second transmembrane domain (148-167), a second extracellular domain (168-184), a third transmembrane domain (185-208), a second intracellular domain (229-223), a fourth transmembrane domain (229-223), and a fourth transmembrane domain (228-230). brand domain (224-244), a third extracellular domain (245-261), a fifth transmembrane domain (262-286), a third intracellular domain (287-309), a sixth transmembrane domain (310-329), a fourth extracellular domain (330-342), a seventh transmembrane domain (343-363) and a C-terminal intracellular domain (364-411).

Although the above CRF2 receptor is provided for purposes of illustration (see also Figure 2 and Sequence I.D. No. 2), the present invention should not be limited thereto. In particular, the present invention contemplates a wide variety of other CRF2 receptors having substantial similarity to the sequences disclosed in Sequences LD. Nos. 1-4. As used herein in the context of the present invention, nucleic acid sequences encoding CRF2 receptors are considered to be substantially similar to those disclosed herein if: (a) the nucleic acid sequence is derived from the coding region of a native CRF2 receptor gene (including, e.g., allelic variants of the sequences disclosed herein); (b) the nucleic acid sequence is capable of hybridizing to nucleic acid sequences of the present invention (or their complementary strands) under conditions of moderate (e.g., 50% formamide, 5 x SSPE, 5 x Denhardt's, 0.1% SDS, 100 µg/ml salmon sperm nucleic acid and a temperature of 42°C) or high stringency (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1989); or (c) nucleic acid sequences that are degenerate as a result of the genetic code for the nucleic acid sequences defined in (a) or (b). Furthermore, although deoxyribonucleic acid molecules are referred to herein, as should be apparent to those skilled in the art from the disclosure provided herein, a wide variety of related nucleic acid molecules may also be used in various embodiments described herein, including, e.g., RNA, nucleic acid analogs, as well as chimeric nucleic acid molecules which may be composed of more than one type of nucleic acid.

In addition, as noted above, "CRF₂ receptors" in the context of the present invention should be understood to include derivatives and analogs of the CRF₂ receptors described above. Such derivatives include allelic variants and genetically engineered variants containing conservative amino acid substitutions and/or small additions, substitutions or deletions of amino acids whose net effect alters the biological activity (e.g., signal transduction) or function of the CRF₂ receptor. Such derivatives are greater than 70 to 75% similar to the corresponding native CRF₂ receptor, preferably greater than 80% to 85% similar, more preferably greater than 90% to 95% similar, and most preferably greater than 97% similar. Percent similarity can be determined, e.g., by comparing sequence information with the GAP program, which uses the alignment procedure of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482, 1981). Briefly, the GAP program defines similarity as the number of aligned symbols (nucleotides or amino acids) that are similar divided by the total number of symbols in the shorter of the two sequences. The preferred deficiency parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides and the weighted comparison matrix of Gribskov and Burgess (Nucl. Acids Res. 14:6745, 1986), as described by Schwartz and Dayhoff (eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979); (2) one penalty point for each gap and another 0.1 penalty point for each symbol in each gap; and (3) no penalty point for gaps at the end.

The primary amino acid structure of CRF2 receptors can also be modified by forming covalent or aggregative conjugates with either chemical moieties such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants. Covalent derivatives can be prepared by attaching specific functional groups to the CRF2R amino acid side chains or at the N- or C-termini. Other derivatives of CRF2R within the scope of the invention include covalent or aggregative conjugates of CRF2R or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugated peptide can be a signal (or leader) polypeptide sequence at the N-terminal region of the protein that co-translationally or post-translationally directs the transfer of the protein from its site of synthesis to its site of function inside or outside the cell membrane or cell wall (e.g., the yeast α-factor leader). CRF2R-protein fusions can also include peptides added to facilitate purification or identification of CRF2R (e.g., poly-His, which allows purification of the protein via, e.g., an NTA-nickel chelating column) or FLAG (Hopp et al., Bio/Technology 6:1204-1210, 1988). Other useful fusion proteins include luciferase, beta-galactosidase (Grey et al., PNAS 79:6598, 1982), trp E (Itakura et al., Science 98:1065, 1977), and protein A (Ublen et al., Gene 23:369, 1983). In preferred embodiments, fusion proteins may include a site that is specifically recognized and cleaved, e.g., by a collagenase (which cleaves x in the sequence Pro-x-Gly-Pro, where x is a neutral amino acid; see Keil et al., FEBS Letters 56:292-296, 1975), or factor Xa (which cleaves after arginine in the sequence Ile-Glu-Gly-Arg; see Nogai et al., Methods Enzymol. 153:461-481, 1987).

The present invention also encompasses CRF₂R proteins with or without associated glycosylation in the native pattern. Briefly, CRF₂R expressed in yeast or mammalian expression systems such as COS-7 cells can be similar or significantly different in molecular weight and glycosylation pattern from the native molecules, depending on the expression system. Expression of CRF₂R nucleic acids in bacteria such as E. coli yields non-glycosylated molecules. Functional mutant analogs of mammalian CRF₂R with inactivated N-glycosylation sites can be prepared by oligonucleotide synthesis and ligation or by site-directed mutagenesis techniques. These analog proteins can be produced in a homogeneous form with reduced carbohydrates in good yield using a yeast expression system. N- Glycosylation sites in eukaryotic proteins are generally characterized by the amino acid triplet Asn-A₁-Z, where A₁ is any amino acid except Pro and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate. Such a site can be eliminated by replacing Asn or the residue Z with another amino acid, by deleting Asn or Z, or by inserting a non-Z amino acid between A₁ and Z or an amino acid other than Asn between Asn and A₁.

Proteins that are biologically active and substantially similar to CRF₂R proteins can also be constructed by, for example, making various substituents of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues can be deleted or replaced by other amino acids to prevent the formation of incorrect intramolecular disulfide bridges upon renaturation. Other approaches to mutagenesis include modifying adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. In general, substitutions should be made conservatively, i.e., the most preferred Replacement amino acids are those with physicochemical properties similar to those of the replaced residues.

If a substitution, deletion or insertion strategy is adopted, the potential effect of the deletion or insertion on biological activity should be considered using, e.g., a binding assay such as that described in the Examples. Particularly preferred regions in which mutations, deletions, substitutions or insertions can be made include those not directly related to the binding of the receptor to its ligand. Such regions include all parts of the receptor except the N-terminal extracellular domain, as well as the third intracellular loop between Th5 and Th6 (see, e.g., Kobilka, Ann. Rev. Neuro. 15:87-114, 1992).

Mutations in nucleotide sequences designed for expression of proteins that are substantially similar to CRF2 receptors should preferably preserve the reading frame phase of the coding sequence. Furthermore, the mutations should preferably not create complementary regions that could hybridize to create secondary mRNA structures, such as loops or hairpins, that would negatively affect the translation of the receptor mRNA. Although a mutation site can be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, to select for optimal properties of mutants at a particular site, random mutagenesis can be performed at a target codon or above a given site and the expressed CRF2 mutants screened for biological activity.

Not all mutations in the nucleotide sequence encoding CRF2R will be expressed in the final product. For example, nucleotide substitutions can be made to enhance expression, primarily to avoid secondary structure loops in the transcribed mRNA, or to provide codons that are more readily translated by the selected host, e.g., the well-known E. coli preference codons for E. coli expression.

Mutations can be introduced at specific sites by synthesizing oligonucleotides containing a mutated sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analogue with the desired amino acid insertion, substitution or deletion.

Alternatively, oligonucleotide-directed site-directed mutagenesis techniques can be used to provide an altered gene with particular codons altered according to the required substitution, deletion or insertion. Typical methods for making the above-identified alterations are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik, Bio Techniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Sambrook et al. (Molecular cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989); and U.S. Patents 4,518,584 and 4,737,462.

CRF2 receptors, as well as substantially similar derivatives or analogs, can be used as therapeutic reagents, immunogens, reagents in receptor-based immunoassays or as binding agents for affinity purification procedures for CRF, sauvagine, urotensin I or other related molecules or other binding ligands such as anti-CRF2 antibodies. In addition, CRF2 receptors of the present invention can be used to screen compounds for CRF2 receptor agonist or antagonist activity. CRF2 receptor proteins can be covalently linked through reactive side groups to various insoluble substrates, such as cyanogen bromine-activated, bisoxirane-activated, carbonyldiimidazole-activated, or tosyl-activated, agarose structures or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-linking). CRF2R, once bound to a substrate, can be used to selectively bind anti-CRF2R antibodies or CRF (for assay or purification purposes).

Isolating CRF₂ receptor DNA clones

As noted above, the present invention provides isolated nucleic acid molecules encoding CRF₂ receptors. Briefly, nucleic acid molecules encoding CRF₂ receptors of the present invention can be readily isolated from a variety of warm-blooded animals, including, for example, humans, macaques, horses, cattle, sheep, pigs, dogs, cats, rats, and mice. Particularly preferred tissues from which nucleic acid molecules can be isolated are Nucleic acid molecules encoding CRF₂ receptors can be isolated include brain and neural tissues such as the hypothalamus, hippocampus and frontal cortex, as well as other tissues such as the lung, heart, skeletal muscle or kidney. Nucleic acid molecules encoding CRF₂ receptors of the present invention can be readily isolated from conventionally prepared cDNA libraries (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1989) or from commercially available libraries (e.g., Stratagene, La Jolla, Calif.) using the disclosure provided herein. Particularly preferred methods for obtaining isolated nucleic acid molecules encoding CRF₂ receptors of the present invention are described in more detail below in Example 1 (see also Sequence ID Nos. 1 and 3).

As noted above, in particularly preferred embodiments of the invention, isolated nucleic acid molecules encoding human CRF₂ receptors are provided. Briefly, such nucleic acid molecules can be readily obtained by probing a human cDNA library with either a specific sequence as described below in Example 1 or with a rat sequence (e.g., Sequence ID Nos. 1 or 3) under low stringency conditions (e.g., 35% formamide, 5 x SSC, 5 x Denharts, 0.1% SDS, 100 ug/ml salmon sperm nucleic acid at 42°C for 12 hours. This can be followed by extensive washing with 2x SSC containing 0.2% SDS at 50°C. Suitable cDNA libraries can be obtained from commercial sources (e.g., Stratagene, La Jolla, Calif.) or prepared using standard techniques (see, e.g., Sambrook et al., supra).

Production of recombinant CRF₂ receptors

As noted above, the present invention also provides recombinant expression vectors comprising synthetic or cDNA-derived nucleic acid fragments encoding CRF₂ receptors or substantially similar proteins operably linked to appropriate transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding appropriate mRNA ribosomal binding sites and, in preferred embodiments, sequences encoding the termination of Control transcription and translation. The ability to replicate in a host, usually conferred by an origin of replication and a selection gene to facilitate recognition of transformants, may additionally be incorporated. Regions of nucleic acid are operably linked if they are functionally related to one another. For example, nucleic acid for a signal peptide (secretory leader) is operably linked to nucleic acid for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. In general, operably linked means adjacent and, in the case of secretory leaders, adjacent and in frame.

Expression vectors may also contain nucleic acid sequences necessary to direct the secretion of a polypeptide of interest. Such nucleic acid sequences may include at least one secretory signal sequence. Typical examples of secretory signals include the alpha factor signal sequence (pre-pro sequence; Kurjan and Herskowitz, Cell 30:933-943), 1982; Kurjan et al., U.S. Patent No. 4,546,082; Brake EP 116,201), the PHO5 signal sequence (Beck et al., WO 86/00637), the BAR1 secretory signal sequence (MacKay et al., US Patent No. 4,613,572; MacKay, WO 87/002670), the SUC2 signal sequence (Carlson et al., Mol. Cell. Biol. 3:439-447), the α-1-antitrypsin signal sequence (Kurachi et al., Proc. Natl. Acad. Sci. USA 78:6826-6830, 1981), and the β-2-plasmin inhibitor signal sequence (Tone et al., J. Biochem. (Tokyo) 102:1033-1042, 1987), the Tissue plasminogen activator signal sequence (Pennica et al., Nature 301:214-221, 1983), the E. coli PhoA signal sequence (Yuan et al., J. Biol. Chem. 265:13528-13552, 1990), or any of the bacterial signal sequences reviewed, for example, by Oliver (Ann. Rev. Microbiol. 39:615-649, 1985). Alternatively, a secretory signal sequence can be synthesized according to the rules given, e.g., by Heinje (Eur. J. Biochem. 133:17-21, 1983; J. Mol. Biol. 184:99-105, 1985; Nuc. Acids Res. 14:4683-4690, 1986).

For expression, a nucleic acid molecule encoding a CRF₂ receptor is inserted into a suitable expression vector, which in turn is used to transform or transfect suitable host cells for expression. Host cells used in the expression Cells used in carrying out the present invention include mammalian, avian, plant, insect, bacterial and fungal cells. Preferred eukaryotic cells include cultured mammalian cell lines (e.g. rodent or human cell lines) and fungal cells, including species of yeast (e.g. Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomyces spp. or Kluyveromyces spp.) or filamentous fungi (e.g. Aspergillus spp., Neurospora spp.). Strains of the yeast Saccharomyces cerevisiae are particularly preferred. Methods for producing recombinant proteins in a variety of prokaryotic and eukaryotic host cells are well known in the art (see "Gene Expression Technology," Method in Enzymology, Volume 185, Goeddel (ed.), Academic Press, San Diego, Calif., 1990; see also "Guide to Yeast Genetics and Molecular Biology," Methods in Enzymology, Guthrie and Fink (eds.) Academic Press, San Diego, Calif., 1991). In general, a host cell will be selected on the basis of its ability to produce the protein of interest at high levels and its ability to carry out at least some of the processing steps required for the protein's biological activity. In this way, the number of cloned nucleic acid sequences that must be transfected into the host cell can be minimized and the overall yield of biologically active protein can be maximized.

Suitable yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1978), YEp13 (Broach et al., Gene 8:121-133, 1979), POT vectors (Kawasaki et al., U.S. Patent No. 4,931,373, which is incorporated herein by reference), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives thereof. Such vectors will generally have a suitable marker, which may be one of a large number of genes having a dominant phenotype for which a phenotypic assay exists to allow transformants to be selected. Preferred markers suitable are those that complement host cell auxotrophy, provide antibiotic resistance, or enable a cell to utilize specific carbon sources, and include LEU2 (Broach et al., supra), URA3, (Botstein et al., Gene 8:17, 1979), HIS3 (Struhl et al., supra), or POT1 (Kawasaki et al., supra). Another suitable marker to select is the CAT gene, which confers chloramphenicol resistance to host cells.

Promoters suitable for use in yeast include promoters of glycolytic yeast genes (Hitzeman et al., J. Biol. Chem. 255:12073-12080, 1980; Alber and Ka wasaki, J. Mol. Appl. Genet. 1:419-434, 1982; Kawasaki, US Patent No. 4,599,311) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al. (ed.), p. 355, Plenum New York, 1982; Ammerer, Meth. Enzymol. 101:192-201, 1983). Particularly preferred promoters in this regard are the TPI1 promoter (Kawasaki, U.S. Patent No. 4,599,311, 1986) and the ADH2-4c promoter (Russell et al., Nature 304:652-654, 1983; Irani and Kilgore, U.S. Patent Application Serial No. 07/784,653, which is incorporated herein by reference). The expression units may also include a transcription terminator, such as the TPI1 terminator (Alber and Kawasaki, supra).

In addition to yeast, proteins of the present invention can be expressed in filamentous fungi, e.g., strains of the fungus Aspergillus (McKnight et al., U.S. Patent No. 4,935,349, which is incorporated herein by reference). Examples of useful promoters include those derived from glycolytic genes of Aspergillus nidulans, such as the ADH3 promoter (McKnight et al., EMBO J. 4:2093-2099, 1985) and the tpiA promoter. An example of a suitable terminator is the ADH3 terminator (McKnight et al., supra 1985). The expression units using such components are cloned into vectors capable of inserting into the chromosomal nucleic acid of Aspergillus.

Techniques for transforming fungi are well known in the literature and have been described, for example, by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75:1929-1933, 1978), Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1740-1747, 1984) and Russell (Nature 301:167-169, 1983). The genotype of the host cell will generally contain a genetic defect that is complemented by the selectable marker located on the expression vector. The selection of a specific host and selectable marker is well within the skill of the art. To optimize production of heterologous proteins in, e.g., yeast, it is preferred that the host strain carries a mutation, such as the pep4 yeast mutation (Jones, Genetics 85:23-33, 1977), which results in reduced proteolytic activity.

In addition to fungal cells, cultured mammalian cells can be used as host cells in the present invention. Preferred cultured mammalian cells for use in the present invention include COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), and 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A preferred BHK cell line is the BHK 570 cell line (deposited with the American Type Culture Collection under accession number CRL 10314). In addition, a number of other mammalian cell lines can be used in the present invention, including rat Hep I (ATCC No. CRL 1600), rat Hep II (ATCC No. CRL 1548), TCMK (ATCC No. CCL 139), human lung (ATCC No. CCL 75.1), human hepatoma (ATCC No. HTB-52), Hep G2 (ATCC No. HB 8065), mouse liver (ATCC No. CCL 29.1), NCTC 1469 (ATCC No. CCL 9.1), SP2/0-Ag14 (ATCC No. 1581), HIT-T15 (ATCC No. CRL 1777), and RINm 5AHT₂B (Orskov and Nielson, FEBS 229(1):175-178, 1988).

Mammalian expression vectors for use in carrying out the present invention should include a promoter capable of directing transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Viral promoters include the cytomegalovirus immediate early promoter (Boshart et al. Cell 41:521-530, 1985) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981). Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., US Patent No. 4,579,821), a mouse Vj promoter (Bergmann et al., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983; Grant et al., Nuc. Acids Res. 15:5496, 1987), and a mouse VH promoter (Loh et al., Cell 33:85-93, 1983). A particularly preferred promoter is the major late promoter of adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-13199, 1982). Such expression vectors may also contain a set of RNA splice sites located downstream of the promoter and upstream of the nucleic acid sequence encoding the peptide or protein of interest. Preferred RNA splice sites may be obtained from SV40 adenovirus and/or immunoglobulin genes. Alternatively, in certain embodiments, RNA splice sites may be located downstream of the nucleic acid sequence encoding the peptide or protein of interest. Also included in the expression vectors is a polyadenylation signal located downstream of the coding sequence of interest. Suitable polyadenylation signals include the early or late polyadenylation signals of SV40 (Kaufman and Sharp, supra), the polyadenylation signal from the adenovirus 5 E1B region, and the terminator of the human growth hormone gene (DeNoto et al., Nuc. Acids Res. 9:3719-3730, 1981). The expression vectors may contain a non-coding viral leader sequence, such as the three partial adenovirus 2 leader located between the promoter and the RNA splice sites. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer and the mouse 1 enhancer (Gillies, Cell 33:717-728, 1983). Expression vectors may also include sequences encoding the adenovirus VA RNAs. Suitable vectors can be obtained from commercial sources (e.g., Invitrogen, San Diego, CA; Stratagene, La Jolla, CA).

Cloned nucleic acid sequences can be introduced into cultured mammalian cells by, e.g., calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978, Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), or DEAE-dextran-mediated transfection (Ausubel et al., (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. NY, 1987), which are incorporated herein by reference. To identify cells that have stably integrated the cloned nucleic acid, generally, a selectable marker is introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs such as neomycin, hygromycin and methotrexate. The selectable marker can be an amplifiable selectable marker. Preferred amplifiable selectable markers are the DHFR gene and the neomycin resistance gene. Selected markers are reviewed in Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA, which is incorporated herein by reference). The selection of selectable markers is within the skill of those skilled in the art.

Selectable markers can be introduced into the cell on a separate vector at the same time as the CRF2 receptor sequence, or they can be introduced on the same vector. When on the same vector, the selectable marker and the CRF2 receptor sequence can be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (e.g., Levinson and Simonsen, U.S. Patent No. 4,713,339). It may also be advantageous to add additional nucleic acids, known as "carrier nucleic acid," to the mixture introduced into the cells.

Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the nucleic acid sequence(s) of interest. Drug selection is then applied to select for growth of cells that stably express the selectable markers. For cells transfected with an amplifiable selectable marker, the drug concentration can be gradually increased to select for increased copy number of the cloned sequences and thereby increase the level of expression. Cells that express the introduced sequences are selected and screened for production of the protein of interest in the desired form or level. Cells that meet these criteria can then be cloned and scaled up for production.

Preferred prokaryotic host cells for use in practicing the present invention include Escherichia coli (e.g., E. coli HB101, E. coli DH1, E. coli MRC1, and E. coli W3110), although Bacillus, Pseudomonas, and Streptomyces and other genera are also useful. Techniques for transforming these hosts and expressing foreign nucleic sequences cloned therein are well known in the art (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982; or Sambrook et al., supra). Vectors used for expression of cloned nucleic acid sequences in bacterial hosts will generally contain a selectable marker, such as a gene for antibiotic resistance, and a promoter that functions in the host cell. Suitable promoters include the trp (Nichols and Yanofsky, Meth. Enzymol. 101:155-164, 1983), lac (Casadaban et al., J. Bacteriol. 143:971-980, 1980), and phage k (Queen, J. Mol. Appl. Genet. 2:1-10, 1983) promoter systems. Plasmids useful for transforming bacteria include pBR322 (Bolivar et al., Gene 2:95-113, 1977), the pUC plasmids (Messing, Meth. Enzymol. 101:20-78, 1983; Vieira and Messing, Gene 19:259-268, 1982), pCQV2 (Queen, supra), pMAL-2 (New England Biolabs, Beverly, MA), and derivatives thereof. Plasmids can contain both viral and bacterial elements.

Based on the teachings herein, promoters, terminators and methods for introducing expression vectors encoding CRF2 receptors of the present invention into plant, avian and insect cells would be apparent to those skilled in the art. The use of, e.g., baculoviruses as vectors for expressing heterologous nucleic acid sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28:215-224, 1990). In addition, the use of Agrobacterium rhizogenes as vectors for gene expression in plant cells has been described by Sinkar et al. (J. Biosci. (Bangalore) 11:47-58, 1987).

Host cells containing nucleic acid molecules of the present invention are then cultured to express a nucleic acid molecule expressing a CRF2 receptor. The cells are cultured in a culture medium according to standard procedures containing nutrients required for the growth of the selected host cells. A variety of suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals as well as other components, e.g., growth factors or serum, required by the specific host cells. The growth medium will generally select cells containing the nucleic acid molecules by, e.g., drug selection or deprivation of an essential nutrient complemented by the selectable marker on the nucleic acid construct or co-transfected with the nucleic acid construct.

Suitable growth conditions for yeast cells include, for example, culturing in a chemically defined medium containing a nitrogen source, which may be a non-amino acid nitrogen source or a yeast extract, inorganic salts, vitamins and essential amino acid supplements at a temperature between 4°C and 37°C, with 30°C being most preferred. The pH of the medium is preferably maintained at a pH greater than 2 and less than 8, preferably pH 5-6. Methods for maintaining a stable pH include buffering and continuous pH control. Preferred agents for pH control include sodium hydroxide. Preferred buffering agents include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, MO). Due to the propensity of yeast host cells to hyperglycosylate heterologous proteins, it may be preferable to express the CRF2 receptors of the present invention in yeast cells defective in a gene required for asparagine-linked glycosylation. Such cells are preferably grown in a medium containing an osmotic stabilizer. A preferred osmotic stabilizer is sorbitol supplemented in the medium at a concentration between 0.1 M and 1.5 M, preferably at 0.5 M or 1.0 M. Cultured mammalian cells are generally cultured in commercially available serum-containing or serum-free media. Selection of a medium and growth conditions appropriate for the particular cell line is within the skill of the art.

CRF2 receptors can also be expressed in non-human transgenic animals, particularly transgenic warm-blooded animals. Methods for producing transgenic animals, including mice, rats, rabbits, sheep, and pigs, are known in the art and are disclosed, for example, in Hammer et al. (Nature 315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983), Brinster et al. (Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985), Palmiter and Brinster (Cell 41:343-345, 1985), and U.S. Patent No. 4,736,866, which are incorporated herein by reference. Briefly, an expression unit containing a nucleic acid sequence to be expressed together with appropriately arranged expression control sequences is introduced into pronuclei of fertilized eggs. Introduction of nucleic acid is usually accomplished by microinjection. Integration of the injected nucleic acid is demonstrated by blot analysis of nucleic acid from tissue samples, typically tail tissue samples. It is generally preferred that the introduced nucleic acid be incorporated into the germ line of the animal so that it is passed on to the animal's offspring.

"Knockout" animals can be developed from embryonic stem cells by the use of homologous recombination (Capecchi, Science 244:1288-1292, 1989) or antisense oligonucleotide (Stein and Chen, Science 261(5124):1004-1012, 1993; Milligan et al., Semin. Conc. Biol. 3(6):391-398, 1992).

A transgenic animal, such as a mouse, can be developed by targeting a mutation to disrupt a CRF2 receptor sequence (see Mansour et al. "Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: A general strategy for targeting mutations to non-selectable genes," Nature 336:348-352, 1988). Such animals can be easily used as a model to study the role of CRF2 receptors in metabolism.

CRF₂ receptor peptides

As noted above, the present invention also provides CRF₂ receptor peptides. In the context of the present invention, the CRF₂ receptor should be understood to include portions of a CRF₂ receptor or derivatives thereof as discussed above which do not contain transmembrane domains and which contain at least 8, and more preferably 10 or more amino acids long. Briefly, the structure of the CRF2 receptor as well as putative transmembrane domains can be predicted from the primary translation products using the hydrophobic plot function of, e.g., P/C Gen or Intelligenetics Suite (Intelligenetics, Mt. View, CA) or according to the procedures described by Kyte and Doolittle (J. Mol. Biol. 157:105-132, 1982). While one does not wish to be bound by a graphical representation based on this hydrophobicity analysis, CRF2 receptors are believed to have the general structure shown in Figures 1 and 2. In particular, these receptors are believed to comprise an extracellular amino terminal domain, three extracellular loop domains, and four intracellular loop domains, each separated by a transmembrane domain.

In one aspect of the invention, isolated CRF₂ receptor peptides are provided which comprise the extracellular amino-terminal domain of a CRF₂ receptor. In a preferred embodiment, an isolated CRF₂ receptor peptide is provided which comprises the amino acid sequence shown in Sequence LD. No. 4 from amino acid 1 to amino acid number 118. In another embodiment, an isolated CRF₂ receptor peptide is provided which comprises the amino acid sequence shown in Sequence I.D. No. 2 from amino acid 1 to amino acid number 138.

CRF2 receptor peptides can be produced by, among other methods, culturing suitable host/vector systems to produce the recombinant translation products of the present invention. Supernatants from such cell lines can then be treated by a variety of purification procedures to isolate the CRF2 receptor peptide. For example, the supernatant can first be concentrated using commercially available protein concentration filters such as an Amicon or Millipore Pellicon ultrafiltration unit. After concentration, the concentrate can be applied to a suitable purification matrix such as, for example, CRF or an anti-CRF2 receptor antibody bound to a suitable support. Alternatively, anion or cation exchange resins can be used to purify the receptor or peptide. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps can be used to further purify the CRF2 receptor peptide.

Alternatively, CRF2 receptor peptides can also be prepared using standard polypeptide synthesis protocols and purified using the procedures described above.

A CRF2 receptor peptide is considered "isolated" or purified in the context of the present invention if only a single band is detected after SDS-polyacrylamide gel analysis followed by staining with Coomassie Brilliant Blue.

Antibodies against CRF₂ receptors

In one aspect of the present invention, CRF2 receptors, including derivatives thereof, as well as portions or fragments of these proteins, such as the CRF2 receptor peptides discussed above, can be used to produce antibodies that specifically bind to CRF2 receptors. In the context of the present invention, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, fragments thereof, such as F(ab')2 and Fab fragments, as well as recombinantly produced binding partners. These binding partners include the variable regions of a gene encoding a specifically binding monoclonal antibody. Antibodies are defined as specifically binding if they bind to the CRF2 receptor with a Ka of greater than or equal to 107 M-1. The affinity of a monoclonal antibody or binding partner can be readily determined by one of skill in the art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949).

Polyclonal antibodies can be readily prepared by one of skill in the art from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats. Briefly, the CRF2 receptor is used to immunize the animal by intraperitoneal, intramuscular, intraocular or subcutaneous injections. The immunogenicity of a CRF2 receptor or CRF2 receptor peptide can be increased by the use of an adjuvant such as complete or incomplete Freund's adjuvant. After several booster immunizations, small serum samples are collected and tested for reactivity with the CRF2 receptor. A variety of tests can be used to detect antibodies that specifically bind to a CRF2 receptor. Example tests are described in detail in Antibodies:A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988.

Typical examples of such assays include countercurrent immunoelectrophoresis (CIEP), radioimmunoassays, radioimmunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, inhibition or competition assays, and sandwich assays (see U.S. Patent Nos. 4,376,110 and 4,486,530; see also Antibodies. A Laboratory Manual, supra). Particularly preferred polyclonal antisera will give a signal at least three times the background. Once the animal's titer has reached a plateau in its reactivity with the CRF2 receptor, larger amounts of polyclonal antisera can be readily obtained either by weekly bled or by bleeding the animal.

Monoclonal antibodies can be readily prepared using well-known techniques (see U.S. Patents RE 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Briefly, in one embodiment, an animal, such as a rat or mouse, is injected with a form of CRF2 receptor that is capable of generating an immune response against the CRF2 receptor. Typical examples of suitable forms include, but are not limited to: Cells expressing the CRF2 receptor or peptides based on the CRF2 receptor sequence. In addition, many techniques are known in the art for increasing the resulting immune response, e.g. by coupling the receptor or receptor peptide to another protein, such as ovalbumin or keyhole limpet hemocyanin (KLH), or by using adjuvants, such as complete or incomplete Freund's adjuvant. The initial immunization can be done by intraperitoneal, intramuscular, intraocular or subcutaneous routes.

Between one and three weeks after the initial immunization, the animal can be reimmunized with another booster immunization. The animal can then be bled for testing and the serum tested for binding to the CRF2 receptor using assays as described above. Additional immunizations can also be performed until the animal reaches a plateau in its reactivity to the CRF2 receptor. The animal can then be given a final booster of CRF2 receptor or CRF2 receptor peptides and sacrificed three to four days later. At this time, the spleen and lymph nodes can be harvested. and dissociated into a single cell suspension by passing the organs through a mesh filter or by disrupting the spleen or lymph node membranes encapsulating the cells. In one embodiment, the red blood cells are subsequently lysed by adding a hypotonic solution followed by immediate return to isotonicity.

In another embodiment, suitable cells for producing monoclonal antibodies are obtained using in vitro immunization techniques. Briefly, an animal is sacrificed and the spleen and lymph node cells removed as described above. A single cell suspension is prepared and the cells are placed in a culture containing a form of the CRF2 receptor capable of generating an immune response as described above. Subsequently, the lymphocytes are harvested and fused as described below.

Cells obtained through the use of in vitro immunization, or from an immunized animal as described above, can be immortalized by transfection with a virus such as Epstein-Barr virus (EBV) (see Glasky and Reading, Hybridoma 8(4):377-389, 1989). Alternatively, in a preferred embodiment, the harvested spleen and/or lymph node cell suspensions are fused with an appropriate myeloma cell to generate a "hybridoma" that secretes monoclonal antibodies. Suitable myeloma lines are preferably defective in antibody construction and expression and, in addition, are syngeneic with the cells of the immunized animal. Many such myeloma cell lines are known in the art and can be obtained from sources such as the American Type Culture Collection (ATCC), Rockville, Maryland (see Catalog of Cell Lines & Hybridomas, 6th ed., ATCC, 1988). Typical myeloma cell lines include: for humans, UC 729-6 (ATCC No. CRL 8061), MC/CAR- Z2 (ATCC No. CRL 8147), and SKO-007 (ATCC No. CRL 8033); for mice, SP2/0-Ag14 (ATCC No. CRL 1581), and P3X63Ag8 (ATCC No. TIB 9); and for rats, Y3-Ag1.2.3 (ATCC No. CRL 1631), and YB2/0 (ATCC No. CRL 1662). Particularly preferred fusion lines include NS-1 (ATCC No. TIB 18) and P3X63 - Ag 8.653 (ATCC No. CRL 1580), which can be used for fusions with either mouse, rat, or human cell lines. The fusion between the myeloma cell line and the cells from the immunized animal can be accomplished by a variety of methods, including the use of polyethylene glycol (PEG) (see Antibodies: A Laboratory Mannual, Harlow and Lane (editors), Cold Spring Harbor Laboratory Press, 1988) or electrofusion (see Zimmermann and Vienken, J. Membrane Biol. 67:165-182, 1982).

After fusion, the cells are placed in culture plates containing an appropriate medium such as RPMI 1640 or DMEM (Dulbecco's modified Eagle's medium) (JRH Biosciences, Lenexa, KS). The medium may also contain additional components such as fetal bovine serum ("FBS", i.e., from Hyclone, Logan, Utah or JRH Biosciences), thymocytes harvested from a pup of the same species used for immunization, or agar to solidify the medium. In addition, the medium should contain a reagent that allows selective growth of fused spleen and myeloma cells. The use of HAT (hypoxanthine, aminopterin and thymidine) (Sigma Chemical Co., St. Louis, MO) is particularly preferred. After about seven days, the resulting fused cells or hybridomas can be screened to determine the presence of antibodies that recognize the CRF2 receptor. After several clonal dilutions and re-testing, a hybridoma can be isolated that produces antibodies that bind to the CRF2 receptor.

Other techniques can also be used to construct monoclonal antibodies (see Huse et al., "Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281, December 1989; see also Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library," Proc. Natl. Acad. Sci. USA 86:5728-5732, August 1989; see also Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas," Strategies in Molecular Biology: 3:1-9, January 1990; these references describe a commercially available system from Stratacyte, La Jolla, California, which allows the production of antibodies by recombinant techniques). Briefly, mRNA is isolated from a B cell population and used to generate cDNA expression libraries of immunoglobulin heavy and light chains in the kIMMUNOZAP(H) and kIMMUNOZAP(L) vectors. These vectors can be screened individually or co-expressed to express Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques can then be converted to non-lytic plasmid, allowing high-level expression of E. coli monoclonal antibody fragments.

Similarly, using recombinant nucleic acid techniques, binding partners can also be constructed to incorporate the variable regions of a gene encoding a specifically binding antibody. The construction of these proteins can be easily carried out by a person skilled in the art (see Lamck et al., "Polymerase Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes From Single Hybridoma Cells", Biotechnology 7:934-938, September 1989; Riechmann et al., "Reshaping Human Antibodies for Therapy", Nature 332:323-327, 1988; Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering", Nature 328:731-734, 1987; Verhoeyen et al., "Reshaping Human Antibodies: Grafting an Antilysozyme Activity", Science 239:1534-1536, 1988; Chaudhary et al., "A Recombinant Immunotoxin Consisting of Two Antibody Variable Domains Fused to Pseudomonas Exotoxin", Nature 339:394-397, 1989, see also, U.S. Patent 5,132,405, entitled "Biosynthetic Antibody Binding Sites") based on the disclosure provided herein. Briefly, in one embodiment, nucleic acid molecules encoding CRF2 receptor-specific antigen binding domains are amplified from hybridomas producing a specifically binding monoclonal antibody and inserted directly into the genome of a cell producing human antibodies (see Verhoeyen et al., supra; see also Reichmann et al., supra). This technique allows the antigen binding site of a specifically binding mouse or rat monoclonal antibody to be converted into a human antibody. Such antibodies are preferred for therapeutic use in humans because they are not antigenic, like the antibodies of rat or mouse.

Alternatively, the antigen binding site (variable region) can be either linked to, or inserted into, another completely different protein (see Chaudhary et al., supra), resulting in a new protein with antigen binding sites of the antibody as well as the functional activity of the other different protein. As one skilled in the art will recognize, the antigen binding sites or CRF2 receptor binding domains of the antibody can be found in the variable region of the antibody. In addition, nucleic acid sequences encoding minor portions of the antibody or variable regions that specifically bind to mammalian CRF2 receptor can also be used in conjunction with the present invention. These portions can be readily tested for binding specificity to the CRF2 receptor using assays described below.

In a preferred embodiment, genes encoding the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using oligonucleotide variable region primers. These primers can be synthesized by one of skill in the art or can be obtained from commercially available sources. Stratacyte (La Jolla, CA) sells mouse and human variable region primers that include, among others, primers for VHa, VHb, VHc, VHd, CH1, VL, and CL regions. These primers can be used to amplify heavy or light chain variable regions, which can then be inserted into vectors such as IMMUNOZAP*(H) and IMMUNOZAP*(L) (Stratacyte), respectively. These vectors can then be introduced into E. coli for expression. Using this technique, large amounts of single-chain protein containing a fusion of the VH and VL domains can be produced (see Bird et al., Science 242:423-426, 1988).

Other "antibodies" may also be prepared using the disclosure provided herein and are thus also considered to fall within the scope of the present invention, including humanized antibodies (e.g., U.S. Patent 4,816,567 and WO 94/10332), microbodies (e.g., WO 94/09817), and transgenic antibodies (e.g., GB 2 272 440).

Once suitable antibodies have been obtained, they can be isolated or purified by many techniques well known to those skilled in the art (see Antibodies: A Laboratory Manual, supra). Suitable techniques include peptide or protein affinity column, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques. In the context of the present invention, the term "isolated" as used to define antibodies or binding partners means "substantially free of other blood components."

Antibodies of the present invention have many applications. For example, antibodies can be used in flow cytometry to sort CRF₂ receptor-bearing cells, or to histochemically stain CRF₂ receptor-bearing tissues. Briefly, to detect CRF₂ receptors on cells, the cells (or tissue) are incubated with a labeled antibody that specifically binds to CRF₂ receptors, followed by detecting the presence of bound antibody. These steps can also be performed with additional steps, such as washings, to remove unbound antibody. Typical examples of suitable labels, as well as methods for conjugating or coupling antibodies to such labels, are described in more detail below.

In addition, purified antibodies can also be used therapeutically to block the binding of CRF or other CRF2 receptor substrates, such as sauvagin or urotensin I, to the CRF2 receptor in vitro or in vivo. Briefly, blocking antibodies are those antibodies that bind to CRF2 receptor epitopes such that CRF is prevented from binding to the receptor or that CRF is prevented from effecting signal transduction. As noted above, a variety of assays can be used to detect antibodies that block or inhibit binding of CRF to the CRF2 receptor, including, inter alia, inhibition and competition assays noted above. In one embodiment, monoclonal antibodies (prepared as described above) are tested for binding to the CRF2 receptor in the absence of CRF, as well as in the presence of various concentrations of CRF. Blocking antibodies are identified as those that, e.g., bind to CRF2 receptors and, in the presence of CRF, block or inhibit binding of CRF to the CRF2 receptor.

Antibodies of the present invention may also be coupled or conjugated to a variety of other compounds for either diagnostic or therapeutic use. Such compounds include, for example, toxic molecules, molecules that are nontoxic but that become toxic upon exposure to a second compound, and radionuclides. Typical examples of such molecules are described in more detail below.

Antibodies to be used therapeutically are preferably provided in a therapeutic composition comprising the antibody or binding partner and a physiologically acceptable carrier or diluent. Suitable carriers or diluents include, among others, neutral buffered saline or saline and may also include additional binding agents or stabilizers such as buffers, sugars such as glucose, sucrose or dextrose, chelating agents such as EDTA, and various preservatives.

Isolation and use of compounds that bind the CRF₂ receptor, including agonists and antagonists

As noted above, the present invention provides a variety of methods for detecting the presence of compounds that bind to CRF2 receptors. For example, in one embodiment, methods for detecting such compounds are provided that comprise the steps of (a) exposing one or more compounds to cells expressing CRF2 receptors under conditions and for a time sufficient to permit binding of the compounds to the receptors and (b) isolating compounds that bind to the receptors so that the presence of a compound that binds to a CRF2 receptor can be detected. As used herein, conditions that permit binding of compounds to cells expressing CRF2 receptors generally range from pH 6.8 to 7.8 and include a suitable buffer such as Tris-HCl or PBS, either with or without bovine serum albumin ("BSA"). Such buffers may also contain magnesium at a concentration of 5 to 20 mM, as well as protease inhibitors such as bacitracin or aprotinin. The sufficient time for binding of the compounds to cells expressing CRF2 receptors generally ranges from 30 to 150 minutes after exposure.

In other aspects of the present invention, methods are provided for detecting the presence of a compound that binds to CRF2 receptors comprising the steps of (a) exposing one or more compounds to an N-terminal extracellular domain of CRF2 receptor under conditions and for a time sufficient to allow binding of a compound to the N-terminal extracellular domain and (b) isolating compounds that bind to the N-terminal extracellular domain of CRF2 receptor so that the presence of a compound that binds to a CRF2 receptor can be detected. Such assays may take a variety of forms including, for example, an enzyme-linked immunosorbent assay, a "sandwich" assay, or the like. In one embodiment, the compounds are labeled with an agent selected from the group consisting of fluorescent molecules, enzymes, and radionuclides.

In addition to assays that detect the presence of compounds that bind to CRF₂ receptors, the present invention also provides methods for detecting both CRF₂ receptor agonists and CRF₂ receptor antagonists exist. In the context of the present invention, CRF₂ receptor agonists should be understood to refer to molecules that are capable of binding to cell surface receptors and thereby stimulating a pathway within the cell. In contrast, CRF₂ receptor antagonists should be understood to refer to molecules that are capable of binding to a CRF₂ receptor but that prevent stimulation, or show greatly reduced stimulation of a pathway within the cell. Various assays can be used to screen or select CRF agonists or antagonists based on the disclosure provided herein.

For example, in one aspect of the present invention, methods are provided for determining whether a selected compound is a CRF2 receptor agonist or antagonist comprising the steps of (a) exposing a selected compound to cells expressing CRF2 receptors under conditions and for a time sufficient to permit binding of the compound and an associated response in terms of intracellular titers of cAMP, and (b) detecting either an increase or decrease in the titer of intracellular cAMP, and thereby determining whether the selected compound is a CRF2 receptor agonist or antagonist.

cAMP can be readily measured using methods well known in the art, including, for example, methods described by Salomon et al (Anal. Biochem. 58:541-548, 1976) or Krishna et al (J. Pharmacol. Exp. Ther. 163:379, 1968) or, preferably, using commercially available kits such as the scintillation neighborhood assay kit from Amersham Corporation or the lzsl-RIA cAMP determination kit from Incstar Corporation (Stillwater, Minnesota). The scintillation neighborhood assay kit measures the production of cAMP by competition of iodinated cAMP with anti-cAMP antibodies.

In further aspects, methods are provided for detecting a CRF₂ receptor agonist or antagonist in a pool of compounds comprising the steps of (a) exposing a pool of compounds to cells expressing CRF₂ receptors under conditions and for a time sufficient to permit binding of the compound and an associated response in terms of intracellular titers of cAMP and (b) isolating compounds which either increase the intracellular titer of cAMP increase or decrease so that the presence of a CRF₂ receptor agonist or antagonist can be detected.

In another aspect, methods are provided for determining whether a selected compound is a CRF2 receptor antagonist comprising the steps of (a) exposing a selected compound in the presence of a CRF2 receptor agonist to a recombinant CRF2 receptor coupled to a pathway under conditions and for a time sufficient to permit binding of the compound to the receptor and an associated response by the pathway, and (b) detecting a reduction in stimulation of the pathway resulting from binding of the compound to the CRF2 receptor relative to stimulation of the pathway by the CRF2 receptor alone, thereby determining the presence of a CRF2 antagonist. In other aspects, methods are provided for determining whether a selected compound is a CRF2 receptor agonist comprising the steps of (a) exposing a selected compound to a recombinant CRF2 receptor coupled to a pathway under conditions and for a time sufficient to permit binding of the compound to a receptor and an associated response through the pathway, and (b) detecting an increase in stimulation of the pathway resulting from binding of the compound to the CRF2 receptor and determining therefrom the presence of a CRF2 receptor agonist.

In particularly preferred embodiments of the inventions, the pathway is a membrane-bound adenylate cyclase pathway and the detecting step comprises measuring the increase or decrease in cAMP production by the membrane-bound adenylate cyclase pathway. Adenylate cyclase activity assays can be performed, e.g., using a method(s) described in the examples below. Generally, such methods measure the level of cAMP stimulation relative to known agonists (e.g., sauvagine, urotensin I or CRF) and generally comprise exposing a cell preparation expressing a biologically active CRF2 receptor to the selected compound in the presence of radiolabeled ATP.

In another embodiment of the invention, the pathway includes a reporter system such as luciferase or β-galactosidase (see Konig et al., Mol and Cell. Neuro. 2:331-337, 1991). For example, in one embodiment of the invention, an expression vector is provided which comprises a cyclic AMP response element, such as a proenkephalin cyclic AMP response element, operably linked to β-galactosidase or luciferase cDNA. The expression vector is then stably transfected into a host cell and the host cell is then transfected with a second expression vector which expresses a CRF2 receptor. Upon activation of the pathway, increased cAMP titers induce expression of the reporter product, such as β-galactosidase or luciferase. If the reporter product is luciferase, it is then exposed to luciferin and photons released during oxidation of luciferin by luciferase are measured.

A variety of compounds can be screened using such methods. Typical examples include blocking antibodies discussed above, CRF2 receptor peptides, and CRF analogs (including both peptide and non-peptide ligands). In addition, a large number of CRF analogues can be generated by saturation mutagenesis of nucleic acid sequences encoding CRF (e.g., Little, Gene 88:143-151, 1989), by segment-oriented mutagenesis (e.g., Shortle et al., Proc. Natl. Acad. Sci. USA 77:5375-5379, 1980), by forced nucleotide misincorporation (e.g., Liao and Wise, Gene 88:107-111, 1990), or by using randomly mutagenized oligonucleotides (Hutchison et al., Proc. Natl. Acad. Sci. USA 83:710-714, 1986). Individual transformants expressing a CRF2 analog can then be cloned or pooled as discussed above.

Other compounds that can also be screened using the methods of the present invention include chemical compounds that are known and are either commercially available (e.g., from Sigma Chemical Co., St. Louis, MO) or readily synthesized by those skilled in the art. In addition, many new compositions provided in combinatorial libraries can also be readily screened using the methods described herein, including, e.g., organic and protein or peptide libraries (see, e.g., Gold and Tuerk, U.S. Patent 5,270,163; and Ladner et al., U.S. Patents 5,096,815, 5,198,346, and 5,223,409).

In a preferred aspect of the invention, screening of chemical libraries (including peptide libraries, libraries of small organic molecules or libraries of compounds derived from combinatorial chemistry) in a high throughput format using the expressed CRF₂ receptors. More specifically, in one embodiment of the invention, a high throughput receptor binding assay can be performed in a 96-well plate and is automated using the BIOMEK 2000 (Beckmann, Fullerton CA). Briefly, 1 x 10⁵ cells transfected with CRF₂ receptors are grown in each well. To this, 0.05 ml of assay buffer (Dulbecco's phosphate buffered saline, 10 mM MgCl₂, 20 µM bacitracin) with or without unlabeled r/hCRF (final concentration 1 µM) is added in duplicate to determine total binding and nonspecific binding. In singlets, 0.05 ml of compounds (mixtures of 1-30 compounds) is added to each well in addition to 0.05 ml of either [125I]-oCRF or [125I]-r/hCRF (final concentration ~200 pM). The mixture is incubated for two hours at 22°C. Since the transfected cells are adherent, a centrifugation step to separate membrane-bound CRF is not required. Next, fluid is aspirated and cells are washed three times with approximately 0.9 ml of PBS. After the third wash and aspiration, 0.2 ml of 4M guanidine thiocyanate is added to each well to solubilize the tissue. An aliquot (0.15 ml) of the solubilized sample is monitored in a γ-counter for radioactivity at approximately 80% efficiency. Compounds showing ≥ 50% inhibition of [125I]CRF binding are tested in a whole dose response competition assay to determine the affinity of these compounds to the CRF2 receptor (Ki value).

Once partially or to homogeneity, as desired, both CRF2 receptor agonists and antagonists can be used therapeutically. In general, substantially pure recombinant CRF antagonists of at least about 50% homogeneity are preferred, with at least about 70% - 80% homogeneity being more preferred and 95% - 99% or more homogeneity being most preferred, particularly for pharmaceutical uses. In general, the antagonists can be administered intradermally ("id"), intracranially ("ic"), intraperitoneally ("ip"), intrathecally ("it"), intravenously ("iv"), subcutaneously ("sc"), intramuscularly ("im"), or directly into a tumor. Typically, the antagonists are present as free bases or acid salts. Suitable salts should be pharmaceutically acceptable. Typical examples include metal salts, alkali and alkaline earth metal salts such as potassium or sodium salts. Other pharmaceutically acceptable salts include citric, succinic, lactic, hydrochloric and hydrobromic acids. Parenteral compositions can be formulated in aqueous isotonic Solutions with a pH between 5.6 and 7.4. Suitable isotonic solutions may include sodium chloride, dextrose, boric acid sodium tartrate and propylene glycol solutions.

Markings

The nucleic acid molecules, antibodies, CRF2 receptors, and CRF2 agonists and antagonists of the present invention can be labeled or conjugated (by either covalent or non-covalent means) to a number of labels or other molecules, including, for example, fluorescent markers, enzyme markers, toxic molecules, molecules that are nontoxic but become toxic upon exposure to a second compound, and radionuclides.

Typical examples of fluorescent labels suitable for use within the present invention include, for example, fluorescein isothiocyanate (FITC), rodamin, Texas red, luciferase, and phycoerythrin (PE). Particularly preferred for use in flow cytometry is FITC, which can be conjugated to the purified antibodies according to the procedure of Keltkamp in "Conjugation of Fluorescein Isothiocyanate to Antibodies I. Experiments on the Conditions of Conjugation," Immunology 18:865- 873, 1970. (See also Keltkamp, "Conjugation of Fluorescein Isothiocyanate to Antibodies. II. A Reproducible Method," Immunology 18:875-8881, 1970; and Goding "Conjugation of Antibodies with Fluorochromes: Modification to the Standard Methods," J. Immunol. Methods 13:215-226, 1970.). For histochemical staining, HRP, which is preferred, can be conjugated to the purified antibodies according to the procedures of Nakane and Kawaoi ("Peroxidase-Labeled Antibody: A New Method of Conjugation," J. Histochem. Cytochem. 22:1084-1091, 1974; see also, Tijssen and Kurstak, "Highly Efficient and Simple Methods for Preparation of Peroxidase and Active Peroxidase Antibody Conjugates for Enzyme Immunoassays," Anal. Biochem. 136:451-457, 1984).

Typical examples of enzyme markers or labels include alkaline phosphatase, horseradish peroxidase, and β-galactosidase. Typical examples of toxic molecules include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A. Typical examples of molecules that are nontoxic but become toxic upon exposure to a second compound include thymidine kinases such as HSVTK and VZVTK. Typical examples of radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99 m, Rh-105, Pd- 109, In-111, I-123, I-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212.

As will be apparent to those skilled in the art in view of the disclosure provided herein, the nucleic acid molecules, antibodies, CRF2 receptors, CRF2 receptor peptides, and CRF2 receptor agonists and antagonists described above can also be labeled with other molecules, such as colloidal gold, as well as with a member of a high affinity binding pair (e.g., avidin-biotin).

Diagnostic use of CRF₂ receptor sequences

In another aspect of the present inventions, probes and primers are provided for detecting CRF2 receptors. In one embodiment of the invention, probes are provided that are capable of hybridizing to CRF2 receptor nucleic acid or RNA. For purposes of the present invention, probes are "capable of hybridizing" to CRF2 receptor nucleic acid if they hybridize to sequences ID. No. 1 or 3 (or their complementary strands) under conditions of moderate or high stringency (see Sambrook et al., supra); but not to CRF receptor nucleic acids. Preferably, the probe can be used to hybridize to appropriate nucleotide sequences in the presence of 50% formamide, 5x SSPE, 5x Denhardt's, 0.1% SDS and 100 ug/ml salmon sperm nucleic acid at 42°C, followed by a first wash with 2x SSC at 42°C and a second wash with 0.2x SSC at 55 to 60°C.

Probes of the present invention can be composed of either deoxyribonucleic acids (DNA), ribonucleic acids (RNA), nucleic acid analogs, or any combination of these, and can be as few as about 12 nucleotides in length, usually about 14 to 18 nucleotides in length, and possibly as long as the entire sequence of the CRF2 receptor. Selecting the probe size is somewhat dependent upon the use of the probe. For example, to detect the presence of various polymorphic forms of the CRF receptor in an individual, a probe that encompasses substantially the entire length of the CRF2 receptor coding sequence is preferred. CRF2 receptor probes can be used to identify polymorphisms linked to the CRF2 receptor gene (see, e.g., Weber, Genomics 7:524-530, 1990 and Weber and May, Amer. J. Hum. Gen. 44:388-396, 1989). Such polymorphisms may be associated with hereditary diseases such as diabetes.

Probes can be constructed and labeled using techniques well known in the art. Short probes of, e.g., 12 or 14 bases can be generated synthetically. Longer probes of about 75 bases or less than 1.5 kb are preferably generated by, e.g., PCR amplification in the presence of labeled precursors such as 32P-dCTP, digoxigenin-dUTP, or biotin-dATP. Probes of greater than 1.5 kb are generally most easily amplified by transfecting a cell with a plasmid containing the relevant probe, growing the transfected cell in bulk, and purifying the relevant sequences from the transfected cells (see Sambrook et al., supra.)

Probes can be labeled by a variety of markers including, e.g., radioactive markers, fluorescent markers, enzymatic markers and chromogenic markers. The use of P is particularly preferred for marking or labeling a specific probe.

Probes of the present invention can also be used to detect the presence of a CRF2 receptor mRNA or nucleic acid in a sample. However, if CRF2 receptors are present in only a limited number, or if it is desired to detect a selected mutated sequence that is present in only a limited number, or if it is desired to clone a CRF2 receptor from a selected warm-blooded animal, it may be advantageous to amplify the relevant sequence so that it can be more easily detected or obtained.

A number of methods can be used to amplify a selected sequence, including, for example, RNA amplification (see Lizardi et al., Bio/Technology 6:1197-1202, 1988; Kramer et al., Nature 339:401-402, 1989; Lomeli et al., Clinical Chem. 35(9):1826-1831, 1989; U.S. Patent 4,786,600), and nucleic acid amplification using polymerase chain reaction ("PCR") (see U.S. Patents 4,683,195, 4,683,202, and 4,800,159) (see also U.S. Patents 4,876,187 and 5,011,769, which disclose a describe an alternative detection/amplification system involving the use of scissile compounds).

In a particularly preferred embodiment, PCR amplification is used to detect or obtain CRF2 receptor nucleic acid. Briefly, as described in more detail below, a nucleic acid sample is denatured at 95°C to generate single-stranded nucleic acid. Specific primers, as discussed above, are then annealed at 37°C to 70°C, depending on the proportion of AT/GC in the primers. The probes are extended at 72°C with Taq polymerase to generate the complementary strand to the template. These steps form a cycle that can be repeated to amplify the selected sequence.

Primers for amplification of a selected sequence should be selected from sequences that are highly specific and form stable duplexes with the target sequence. The primers should also be non-complementary, particularly at the 3' end, they should not form dimers with themselves and other primers, and they should not form secondary structures and duplexes with other regions of nucleic acids. In general, primers of about 18 to 20 nucleotides are preferred and can be easily synthesized using techniques well known in the art.

Therapeutic uses of CRF₂ receptor(s) and CRF receptor antagonists

CRF2 receptors (or parts thereof), CRF2 receptor agonists and antagonists, as well as nucleic acid sequences encoding these molecules, can be used in a variety of therapeutic applications. For example, CRF2 receptors (or parts thereof that bind to a substrate such as CRF, sauvagin or urotensin I) can be used to reduce vascular titers of the substrate or high ACTH titers resulting from excess of the substrate. Thus, CRF2 receptors can be used in treating disorders associated with high levels of cortisol, e.g., Cushing's disease, alcoholism, anorexia nervosa and other related disorders).

Similarly, CRF₂ receptors or portions thereof that bind to a substrate as discussed above can be used in treating tumors that produce high titers of a substrate (e.g., CRF), such as pituitary tumors, as well as in treating abnormalities during pregnancy, such as preeclampsia, that are associated with elevated CRF titers. Similarly, they can be used to treat hypotension, to modulate the action of immune system disorders, such as arthritis, and to modulate the action of pituitary disorders. In addition, CRF₂ receptors (or portions thereof) to modulate a variety of brain functions, including e.g. control of satiety, reproductive growth, anxiety, depression, fever, metabolism.

In another aspect of the present invention, viral vectors are provided that can be used to treat diseases where either the CRF2 receptor (or a mutant CRF2 receptor) is overexpressed, or where no CRF2 receptor is expressed. Briefly, in one embodiment of the invention, viral vectors are provided that direct the function of antisense CRF2 receptor RNA to prevent overexpression of CRF2 receptors or expression of mutant CRF2 receptors. In another embodiment, viral vectors are provided that direct the expression of CRF2 receptor cDNA. Viral vectors suitable for use in the present invention include, among others, herpes virus vectors (e.g., U.S. Patent 5,288,641), retroviruses (e.g., EP 0 415 731; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent 5,219,740; WO 93/11230; WO 93/10218; Vile and Hart, Cancer Res. 53:3860-3864, 1993, Vile and Hart, Cancer Res. 53:962-967; 1993 Ram et al.; Cancer Res. 53:83-88, 1993; Takamiya et al. J. Neurosci. Res 33: 493-503, 1992; Baba et al., J. Neurosurg 79. 729-735, 1993), pseudotyped viruses, adenoviral vectors (e.g. WO 94/26914, WO93/9191; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS 90 (24):1149 8-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-1207, 1993; 75(2):207-216, 1993; Li et al., Hum. Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur.J. Neurosci. 5(10:1287-1291, 1993; Vincent et al., Nat. Genet. 5(2):130-134, 1993; Jaffe et al., Nat. Genet. 1(5):372-378, 1992; and Levrero et al. Gene 101 (2):195-202, 1991), adenovirus-associated viral vectors (Flotte et al., PNAS 90(22):10613-10617, 1993), parvovirus vectors (Koering et al., Hum. Gene Therap. 5:457-463, 1994), baculovirus vectors, and poxvirus vectors (Panicali and Paoletti, PNAS 79:4927-4931, 1982; and Ozaki et al., Biochem. Biohys. Res. Comm. 193(2):653-660, 1993). In various embodiments, either the viral vector itself or a viral particle containing the viral vector can be used in the methods and compositions described below.

In other embodiments of the invention, the vectors containing or expressing the nucleic acid molecules of the present invention or the nucleic acid molecules

itself, can be administered by a variety of alternative techniques, including, for example, direct nucleic acid injection (Acsadi et al., Nature 352:815-818, 1991); liposomes (Pickering et al., Circ. 89(1):13-21, 1994; and Wang et al., PNAS 84:7851-7855, 1987); lipofection (Felgner et al., Prod. Natl. Acad. Sci. USA 84:7413-7417, 1989); microprojectile bombardment (Williams et al., PNAS 88:2726-2730, 1991); Nucleic acid ligand (Wu et al., J. of Biol. Chem. 264:16985-16987, 1989); administration of nucleic acid coupled to killed adenovirus (Michael et al., J. Biol. Chem. 268(10):6866-6869, 1993; and Curiel et al., Hum. Gene Ther. 3(2):147-154, 1992), retrotransposons, cytofectin-mediated delivery (DMRIE-DOPE, Vical, Calif.), and transferrin-nucleic acid complexes (Zenke).

Improve learning and memory

As noted above, methods are disclosed for improving learning and memory by administering to the patient a therapeutically effective amount of a CRF2 receptor antagonist. Briefly, such patients can be identified by a clinical diagnosis based on symptoms of dementia or learning and memory loss. For example, patients with an amnesic disorder are impaired in their ability to learn new information or are unable to recall recently learned information or past events. The memory deficit is most evident in tasks requiring spontaneous recall and may also be evident when the examiner provides stimuli for the person to recall at a later time. The memory impairment must be sufficiently severe to cause marked impairment in social and occupational functioning and must represent a substantial decline from a previous measure of functioning.

Dementia is characterized by multiple clinically significant cognitive defects that represent a substantial change from a previous measure of functioning. Memory impairment, which includes the inability to learn new material or forgetting recently learned material, is required to diagnose dementia. Memory can be formally tested by asking the person to attend to, retain, recall, and recognize information. The diagnosis of dementia also requires at least one of the following cognitive impairments: aphasia, apraxia, agnosia, or a disorder of executive function. These deficits in language, motor performance, object recognition, or abstract thinking must be considered in conjunction with the Memory deficits must be sufficiently severe to cause impairment in occupational and social functioning and must represent a reduction from a previously higher level of functioning.

A standard test used by clinicians to determine whether a patient has learning and memory impairment is the Mini-Mental Test of Learning and Memory (Folstein et al., J. Psychiatric Res. 12:185, 1975). This test includes a number of simple tasks and written questions. For example, "paired-associated" learning ability is impaired in several types of amnesic patients, including those suffering from head trauma, Korsakoff's disease, or stroke (Squire, 1987). Ten pairs of unrelated words (e.g., army table) are read to the subject. Subjects are then asked to recall the second word when the first word of each pair is given. The measure of memory impairment is a reduced number of paired-associated words recalled relative to a matched control group. This serves as an index of short-term, working memory of the kind that deteriorates in the early stages of dementia or amnesic disorders.

Improvement in learning and memory constitutes either (a) a statistically significant difference between the performance of patients treated with CRF2 receptor antagonists compared with members of a placebo group; or (b) a statistically significant change in performance toward normality on measures appropriate to the disease model. Animal models or clinical cases of disease exhibit symptoms that are by definition distinguishable from normal controls. Thus, the measure of effective pharmacotherapy will be a significant, but not necessarily complete, reversal of symptoms. Improvement can be facilitated in both animal and human models of memory pathology by clinically effective "cognition-enhancing" agents designed to improve performance on a memory task. For example, cognitive enhancers acting as cholinomimetic replacement therapies in patients suffering from dementia and Alzheimer-type memory loss can significantly improve short-term working memory in such paradigms as the paired-association task (Davidson and Stern, 1991). Another potential application for therapeutic interventions against memory impairment is suggested by age-related performance deficits that can be effectively addressed by the Longitudinal study of recent memory in aging mice (Forster and Lal, 1992).

In animals, several established models of learning and memory are available to examine the beneficial cognitive enhancement effects and potential anxiety-related side effects of activating CRF-sensitive neurons. For example, both the Morris maze (Stewart and Morris, in Behavioral Neuscience, R. Saghal, ed. (IRL Press, 1993), p. 107) and the Y-maze (Brits et al., Brain Res. Bull. 6, 71 (1981)) tests measure cognitive enhancement effects. Anxiety-related effects can be assessed in the elevated plus maze (Pellow et al., J. Neurosci. Meth. 14:149, 1985).

In short, the Morris water maze is one of the best validated models of learning and memory, and it is sensitive to the cognitive enhancement effects of a variety of pharmacological agents (McNamara and Skelton, Brain Res. Rev. 18:33, 1993). The task performed in the maze is particularly sensitive to manipulation of the hippocampus in the brain, an area of the brain important for spatial learning in animals and memory consolidation in humans. Furthermore, improvement in performance in the Morris water maze is predictive of the clinical effectiveness of a compound as a cognitive enhancer. For example, treatment with cholinesterase inhibitors or selective muscarinic cholinergic agonists reverses learning defects in the Morris maze animal model of learning and memory, as well as in clinical populations with dementia (McNamara and Skelton, 1993; Davidson and Stern, 1991; McEntee and Crook, 1992; Dawson et al., 1992). In addition, this animal paradigm accurately models the increasing degree of weakness with age (Levy et al., 1994) and the increasing vulnerability of the memory pathway to pre-test delay or interference (Stewart and Morris, 1993) that characterize amnesic patients.

The test is a simple spatial learning task in which the animal is placed in lukewarm water that is opaque due to the addition of powdered milk. The animals learn the location of the platform relative to visual cues placed within the maze and the test room; this learning is referred to as place learning. Briefly, groups of animals receive 1 to 3 ICV injections of control solution or 0.1, 1, 5, or 25 μg of a CRF2 receptor antagonist 15 minutes before training on each of the days.

Control animals typically reach the platform within 5 to 10 seconds after 3 days of training. The measure of the memory-modulating effects of a CRF2 receptor antagonist is a shift in this time period.

The Y-maze test, based on visual discrimination, is another test of learning and memory in animals. In this maze, two arms of the maze end in a transparent plastic panel behind which is a 40-watt electric bulb. The exit box is separated from the third arm by a manually operated trapdoor. In the first trial, all animals are allowed to explore the maze for five minutes and food grains are available in each arm. On the second day, each animal is placed in the start box with the door closed. When the door is opened, the animal is allowed to walk down the arms and eat the grains contained in both arms. On the third day, the animals are given six trials in groups of three with one arm closed at the decision point, no discriminatory stimulus present, and two food grains available in the open goal box. On days four to ten, a light is switched on at the end of the arm containing the food grains and ten trials are carried out, again in groups of three. The time it takes the animal to reach the food grains is recorded.

The efficacy of a CRF2 receptor antagonist to enhance learning and memory in the Y-maze is tested as follows. Fifteen minutes before each of these experimental training blocks on days 4 through 10, groups of animals receive ICV injections of control solutions or doses containing 1, 5, or 25 µg of a CRF2 receptor antagonist. Control animals are expected to make 50% correct choices. The measure of treatment efficacy on memory is an increase in correct responses.

The elevated plus maze test measures anxiogenic responses in an approach-avoidance situation involving an exposed, lighted room versus a dark, enclosed room. Both rooms are elevated and set up as two running tracks that intersect in the shape of a plus sign. This type of approach-avoidance situation is a classic text for "emotionality" and is very sensitive to treatments that produce disinhibition and stress. Briefly, animals are placed in the center of the maze and allowed free access to all four arms in a 5-minute test period. The time spent in each arm is recorded.

In humans, the determination of improvement in learning and memory can be measured by such tests as the Wechsler Memory Scale or a paired-associated memory task. The Wechsler Memory Scale is a widely used pencil-and-paper test of cognitive function and memory performance. In the normal population, the standard test yields a mean of 100 and a standard deviation of 15, so that mild amnesia can be detected with a 10 to 15 point reduction in the score, or more severe amnesia with a 20 to 30 point reduction, etc. (Squire, 1987). During the clinical interview, a battery of tests including, but not limited to, the Mini-Mental Test, the Wechsler Memory Scale or paired-associated learning are administered to diagnose symptomatic memory loss. The tests provide general sensitivity to general cognitive impairment and specific loss of learning/memory capacity (Squire, 1987). In addition to specifically diagnosing dementia or amnesic disorders, these clinical instruments also identify age-related cognitive decline, which reflects an objective reduction in mental function due to the aging process that is within normal limits given the age of the person (DSM IV, 1994). As stated above, "improvement" in learning and memory in the context of the present invention is present when there are statistically significant differences toward normality on the paired-associated test between, for example, the performance of CRF2 receptor antagonist- treated patients compared with members of the placebo group or between subsequent tests administered to the same patient.

Alzheimer's disease

Methods are disclosed for treating Alzheimer's disease (AD) by administering to a patient a therapeutically effective amount of a CRF2 receptor antagonist. Briefly, such patients can be identified by clinical diagnosis based on symptoms of dementia or learning and memory loss that cannot be attributed to other causes. In addition, these patients are also identified by diagnosis of brain atrophy as determined by magnetic resonance imaging.

Several established animal models of Alzheimer's disease that focus on cholinergic defects are available. The primary role of cholinergic defects in AD is well established. In AD, there are significant positive correlations between reduced choline acetyltransferase activity and reduced CRF titers in the frontal, occipital, and temporal lobes (DeSouza et al., 1986). Similarly, there is negative correlation between reduced choline acetyltransferase activity and increased numbers of CRF receptors in these three cortices (Id:). There is, in two other neurodegenerative diseases, a highly significant correlation between CRF and choline acetyltransferase activity in Parkinson's disease, but only a slight correlation in progressive supranuclear palsy (Whitehouse et al., 1987).

In rats, anatomical and behavioral studies demonstrate interactions between CRF and cholinergic systems. First, CRF and acetylcholinesterase are co-localized in some brain stem nuclei, and some cholinergic neurons also contain CRF. Second, CRF inhibits carbachol-induced behaviors (carbachol is a muscarinic cholinergic receptor antagonist), suggesting that CRF has effects on cholinergic systems (Crawley et al., Peptides 6:891, 1985). Treatment with another muscarinic cholinergic receptor antagonist, atropine, results in an increase in CRF receptors (De Souza and Battaglia, Brain Res. 397:401, 1986). Taken together, these data demonstrate that CRF and cholinergic systems interact in a similar manner in humans and in animals.

An animal model of Alzheimer's disease that targets cholinergic deficiencies is produced by administration of scopolamine, a non-selective postsynaptic muscarinic receptor antagonist that blocks stimulation of postsynaptic receptors by acetylcholine. In these animals, memory deficits are readily apparent as measured by passive avoidance tests or delayed-location-mapping tests that distinguish motor or perceptual deficits from amnesia or cognitive enhancement effects from experimental treatments. Thus, Morris maze and Y-maze tests are used after scopolamine-induced amnesia to test memory impairment and subsequent enhancement following administration of a CRF2 receptor antagonist. In the Morns Maze, the design of the test is essentially as described above, but is modified to include treatment 30 minutes before exercise on each of days one through three with an ip injection of scopolamine hydrobromide (0.3 mg/kg). This amnesic dose of scopolamine attenuates the er acquisition and retention of spatial and avoidance learning paradigms in the rat. The anti-amnesic effects of 1, 5, or 25 µg of a CRF₂ receptor antagonist are measured relative to matched control groups receiving or not receiving scopolamine. The effect of the CRF₂ receptor antagonist in reversing scopolamine-induced amnesia using the Y-maze is performed in a manner similar to the Y-maze test described above. Modification of this test includes treatment 30 minutes prior to training on days 5 to 10 with an ip injection of scopolamine hydrobromide (0.3 mg/kg). The anti-amnesic effects of 1, 5, or 25 μg of a CRF2 receptor antagonist administered ICV, centrally or systemically, are measured relative to parallel control and scopolamine-treated control groups.

Several tests that measure cognitive behavior in AD have also been constructed (see Gershon et al., Clinical Evaluation of Psychotropic Drugs: Principles and Guidelines, Prien and Robinson (eds.), Raven Press, Ltd., New York, 1994, p. 467). One of these tests, BCRS, measures concentration, recent memory, past memory, orientation and functioning, and self-protection. The BCRS is designed to measure only cognitive functions. This test, as well as the Weschler Memory Scale and the Alzheimer's Disease Associated Scale, can be used to determine improvement after therapeutic treatment with a CRF2 receptor antagonist. As noted above, "improvement" in Alzheimer's disease in the context of the present invention is present when there is a statistically significant difference toward normality in the Weschler Memory Scale test between, e.g., the performance of patients treated with CRF2 receptor antagonists compared with members of the placebo group or between subsequent tests performed on the same patient. In addition, scopolamine-induced amnesia in humans can be used as a model system to test the efficacy of CRF2 receptor antagonists.

Cerebrovascular disease

As noted above, methods are disclosed for treating cerebrovascular diseases such as stroke, reperfusion injury and migraine by administering to a patient a therapeutically effective amount of a CRF₂ receptor antagonist. A patient is considered to be treated when administration of the CRF₂ receptor antagonists results in a statistically significant improvement, compared with controls, of a clinical or diagnostic indication of cerebrovascular disease (see, e.g., Harrison's Principles of Internal Medicine, McGraw-Hill Book Co.). Typical examples of animal model systems to test the effects of a CRF2 receptor antagonist on cerebrovascular disease include focal ischemic damage from injectables of NMDA (1-aminocyclobutane-cis-1,3-dicarboxylic acid) agonists. Such excitotoxic compounds can be infused into specific areas of the brain and toxicity measured by subsequent staining with a compound such as tetrazolium. Evidence of neural protection can be demonstrated by reduction in the volume of infarction of animals treated with a CRF2 receptor antagonist.

Administration of CRF2 receptor antagonists may be accomplished by a variety of routes, including, for example, by direct injection into a site of injury or disease, by other intracranial, intradermal, intraperitoneal, intrathecal, subcutaneous or intramuscular routes, or more preferably orally or intravenously.

The following examples are provided for purposes of illustration and not for purposes of limitation.

EXAMPLES EXAMPLE 1 Isolation of CRF₂ receptor cDNA A. Isolation of CRF2 receptor cDNA from a rat brain cDNA library.

Male Sprague-Dawley rats (Madison, WI) weighing between 175-250 g are decapitated and the brain is excised. Total RNA is then isolated from the brain using a Promega RNAnts Total RNA Kit (catalog no. Z5110, Promega, Wisc.) according to the manufacturer's instructions, followed by isolation of a poly A+ RNA using a Promega PolyATract kit (catalog no. Z5420). A cDNA phage library is then constructed using a Giga-Pack Gold library construction kit according to the manufacturer's instructions (catalog no. 237611, Stratagene, La Jolla, Calif.), which is again plated and screened essentially as described. described by Sambrook et al., (Molecular Cloning) with oligonucleotide 5'-CCCGGATGCC TACAGAGAAT GCCTGGAGGA TGGGACCTGG GCCTCAAGGG-3" (Sequence ID No. 5). This oligonucleotide is complementary to nucleotides 440-490 of the rat CRF₂ receptor cDNA sequence shown in Fig. 1.

The phage library is rescreened until a single pure phage isolate is obtained. The phage is then grown on the bacterial host XL1-Blue (Stratagene, La Jolla, Calif.) and plasmid nucleic acid is excised into SOLR cells using ExAssist helper phage (Stratagene). The SOLR cells are then plated, plasmid nucleic acid is isolated and sequenced using the Sanger dideoxy protocol.

A rat CRF2 receptor cDNA sequence that can be obtained using this procedure is given below in Figure 2 and in Sequence I.D. No. 1.

B. Isolation of CRF2 receptor cDNA from a rat hypothalamus cDNA library

CRF2 receptor cDNA can also be isolated from commercially available rat hypothalamus cDNA libraries. Briefly, two million plaques are plated from a rat hypothalamus phage library (Stratagen, catalog no. 936518) according to the manufacturer's instructions and screened with oligonucleotide sequence ID No. 5 essentially as described above.

A cDNA sequence for rat CRF2 receptor that can be obtained using this procedure is given below in Figure 1 and in Sequence I.D. No. 3.

C. Isolating CRF₂ receptor cDNA from human cDNA library

CRF2 receptor cDNA can also be isolated from commercially available human cDNA libraries. Briefly, approximately 2 million plaques from a phage library of human frontal cortex (Stratagene, catalog no. 936212) are plated according to the manufacturer's instructions and screened with oligonucleotide 5'-GATCAACTAC TCACAGTGTG AGCCCATTTT GGATGACAAG CAGAGGAAGT A-3' (Sequence I.D. No. 6) as essentially described above.

A cDNA sequence of human CRF2 receptor that can be obtained using this procedure is given below in Sequence I.D. No. 7. Briefly, the human sequence is 89.1% identical at the nucleotide level and 93% identical at the amino acid level to that described above for rat CRF2α receptor (Sequence I.D. No. 3).

EXAMPLE 2 Expression of CRF₂ receptor cDNA

pCDM7-Amp is first constructed from pCDM8 (Seed, Nature 329:840-842, 1987; Seed and Aruffo, Proc. Natl. Acad. Sci. 84:3365-3369, 1987; Thomsen et al., Cell 63:485-493, 1990; Bernot and Auffray, Proc. Natl. Acad. Sci. 88:2550-2554, 1991; Han et al., Nature 349:697-700, 1991) by deletion of the adeno replication origin, M13 replication origin, and sup F selection marker. An ampicillin resistance marker is added to facilitate selection of the plasmid.

A full-length CRF2 receptor clone in pBluescriptSK- is isolated from the phage clone described above and cut with EcoRV and XhoI, resulting in release of the insert. The insert is then isolated and ligated into pCDM7-Amp, which has been cut in a similar manner. The resulting product is used to transform E. coli DH5α, from which larger amounts of plasmid nucleic acid can be isolated.

COS-7 (ATCC No. CRL 1651) cells are then transfected with pCDM7-Amp containing CRF2 receptor cDNA (10 µg nucleic acid/10 cm plate of cells) using 400 µg/ml DEAE-dextran and 100 µM chloroquine. Cells are transfected for four hours, then shocked with 10% DMSO for two minutes. Cells are then washed and grown in DMEM containing 10% fetal bovine serum for two days in a 24-well plate.

EXAMPLE 3 CRF₂ receptor binding assay materials

125I-Tyr-0-ovine-CRF (125I-oCRF; specific activity 2200 Ci/mmol) is obtained from Du Pont-New England Nuclear (Boston, MA). Unlabeled rat/human CRF is purchased from Peninsula Laboratories (Belmont, CA). All other standard reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

A. Cell membrane production

Transfected cells expressing CRF2 receptors are harvested, washed once in PBS (pH 7.0 at 22°C), pelleted by centrifugation, and stored at -70°C until use. On the day of assay, frozen pellets are resuspended in 5 ml of ice-cold buffer containing 50 mM Tris, 10 mM MgCl2, 2 mM EGTA pH 7.0 at 22°C and homogenized using a Polytron (PT3000, Brinkmann Instruments, Westbury, N.Y.) at 27,000 rpm for 20 seconds. The homogenate is centrifuged at 48,000 x g for 10 minutes at 4ºC, the supernatant is discarded and the pellet is rehomogenized in the same volume of buffer and centrifuged again at 48,000 x g for 10 minutes at 4ºC. The resulting pellet containing membranes is resuspended in the above buffer to give a final protein concentration of 300 mg/ml for use in the assay described below. Protein determinations were made according to the procedure of Lowry et al. (J. Biol. Chem. 193:265-275, 1951) using bovine serum albumin (BSA) as a standard.

B. CRF receptor binding assay

100 microliters of the membrane suspension are added to 1.5 ml (polypropylene microfuge tubes) containing 100 μl (100-200 pM) 125I-oCRF in incubation buffer (50 mM Tris, 10 mM MgCl2, 2 mM EGTA, 1.5% BSA, 0.15 mM bacitracin and 1.5% aprotinin) and 100 μl of the incubation buffer. Competing peptides (e.g., r/hCRF, sauvagin, urotensin I, urotensin II, bovine CRF (b-CRF) and deamidated bovine CRF (b-CRF-OH)) are also added. Incubations are carried out at room temperature (22ºC) for 2 hours and terminated by centrifugation in a microfuge for 5 minutes at 12,000 x g. The resulting pellet is gently washed with 1.0 ml of ice-cold phosphate buffered saline, pH 7.2, containing 0.01% Triton X-100 and recentrifuged for 5 minutes at 12,000 x g. The microfuge tubes are cut just above the pellet, placed in 12 x 75 mm polystyrene tubes and monitored for radioactivity in a Packard Cobra II gamma counter at an efficiency of approximately 80%. Nonspecific binding of 125I-oCRF to membrane homogenates is defined in the presence of 1 mM unlabeled CRF.

C. Saturation curve analysis

For saturation studies, 100 μl of 125I-oCRF (50 pM - 10 nM final concentration), 100 μl of assay buffer (with or without 1 μM r/hCRF final concentration to define non-specific binding) and 100 μl of membrane suspension (as described above) are added in order to 1.5 mL polypropylene microfuge tubes. All reactions are run for 2 hours at 22ºC and terminated by centrifugation for 5 minutes at 12,000 x g. Aliquots of the supernatant are collected to determine the amount of unbound radioligand. The remaining supernatant is aspirated. The pellets are gently washed with ice-cold PBS plus 0.01% Triton X-100, recentrifuged and monitored for bound radioactivity. Data from saturation curves are analyzed using the nonlinear least squares curve fitting program LIGAND (Munson and Rodbard, Anal. Biochem 107:220-229, 1980). Nonspecific binding is not arbitrarily defined by the investigators but is estimated as an independent variable from the entire data set.

D. Competition curve analysis

For competition studies, 100 μl of 125I-oCRF (final concentration 200-300 pM) is incubated together with 100 μl of buffer (in the presence of 1 pM to 10 mM competing ligand) and 100 μl of membrane suspension prepared as above. The reaction is allowed to proceed for 2 hours at 22ºC and terminated by centrifugation as described above. Data from competition curves were also analyzed by the LIGAND program. For each competition curve, estimates for the affinity of the radio labeled ligands for the CRF receptor (125I-oCRF or 125Ir/hCRF) in independent saturation experiments. These estimates are constrained during the analysis of the apparent inhibition constants (Ki) for the various related and unrelated peptides.

Routinely, data are analyzed using a one- or two-site model by comparing the "goodness of fit" between the models to accurately determine the Ki. Statistical analyses provided by LIGAND allowed the determination of whether a single-site or multiple-site model should be used. For CRF-related peptides, all data fit a single-site model, suggesting that the transfected cells contained a single homogeneous population of high-affinity binding sites with the appropriate pharmacological profile of the CRF receptor.

EXAMPLE 4 cAMP test

The effect of CRF and related peptides on adenylate cyclase activity in transfected cells can be determined essentially as follows. For agonist testing, compounds are added to wells of buffer alone to identify compounds with intrinsic activity. For antagonist testing, compounds are added to the wells along with 1-100 nM CRF to stimulate the adenylate cyclase system. Compounds are then evaluated for their ability to inhibit CRD-stimulated cAMP production in the transfected cells. Briefly, cells are plated and grown in DMEM containing 10% fetal bovine serum for 2 to 4 days in 24-well plates at 37°C. On the day of the assay, the medium is removed by vacuum aspiration and 100 µl of DMEM, 10 mM MgCl2, 1 mM isobutylmethylxanthine (a phosphodiesterase inhibitor to inhibit degradation of cAMP produced) and 0.1% BSA (pH 7.2) are added to each well. CRF, related peptides and organic test molecules are added to individual wells. Plates are incubated at 37ºC for 1 hour. After incubation, wells are aspirated, rinsed once with PBS and aspirated again. 300 µl of 95% ethanol and 20 mM HCl (EtOH/HCl) are then added to each well and plates are incubated at -20ºC overnight to extract the cAMP produced. The EtOH/HCl is removed, in 1.5 ml polypropylene Eppendorf tubes and the wells were washed with an additional 200 μl of EtOH/HCl and combined with the original sample. All samples were dried in a Speed-Vac (Savant Instruments, Farmingdale NY) and either stored at -20ºC until use or reconstituted with 500 μl of sodium acetate buffer pH 7.5 and immediately assayed for cAMP concentration using a radioimmunoassay kit from Biomedical Technologies Inc. (Stoughton MA) according to the manufacturer's instructions.

Figure 3 depicts cAMP accumulation in cells transfected with the CRF1 receptor and stimulated with ovine CRF (oCRF), rat/human CRF (r/hCRF), sauvagine, or urotensin I. Briefly, this graph shows a dose-dependent increase in cAMP in response to these compounds. All of these compounds show similar potencies. In Figure 4, the rat CRF2α receptor has been transfected into cells and cAMP accumulation is measured in response to the same drugs as shown in Figure 3. As shown in Figure 4, sauvagine and urotensin I are more potent in stimulating cAMP than either oCRF or r/hCRF. Together, these results demonstrate a distinctly different pharmacological profile between CRF1 and CRF2 receptors.

Figures 5 and 6 depict the effect of the antagonists α-helical oRCF(9-41) (see Rivier et al., Science224:889-891, 1984) and d-Phe r/hCRF(12-41) in CRF1- and CRF2α-transfected cells, respectively, after sauvagin-stimulated cAMP production (see Rivier et al., J. Med. Chem. 36:2851-2859, 1993).

The above results have been summarized in Table I below, with the effective concentration for half-maximal adenylate cyclase stimulation (EC50) indicated. TABLE I

EXAMPLE 5 TISSUE DISTRIBUTION OF CRF₂α- AND β-RECEPTORS

To determine the anatomical distribution of the two CRF2 receptor forms, mRNA expression patterns were analyzed in isolated RNA (RNase protection assays) and whole tissue sections (in situ hyperization) of rat brain and peripheral tissues, essentially as described below.

A. RNase Protection Test

A 366-base riboprobe containing a 258-base antisense sequence specific for the rat CRF2α receptor and a 278-base riboprobe containing a 181-base antisense sequence specific for the CRF2β receptor mRNA were generated by T3 RNA polymerase in 40 mM Tris-HCl (pH 7.5), 6 mM MgCl, 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 1 U/ul RNasin (Promega), 0.5 mM each of ATP, CTP, TTP and 3 µM 32P-UTP (DuPont, 800 Ci/mmol), 0.1 µg/ul template DNA, 1 U/ul T3 RNA polymerase in a total volume of 10 µl at 37°C for 30 minutes. A 712-base riboprobe containing 632 bases of antisense sequence specific for rat β-actin mRNA was prepared under the same conditions except that T7 RNA polymerase was used. The probe synthesis mixtures were then treated with DNase I at a concentration of 1 U/ul at 37°C for 10 minutes and then electrophoresed on a denaturing 5% acrylamide gel. The 32P-labeled RNA probes with the expected sizes were recovered from the gel using an RNA purification kit, RNaidkitt (Bio-101, La Jolla, CA). RNA hybridization, RNase digestion, and separation of the protected RNAs was performed as described by Sawbrook et al., above. In assays for CRF2 receptor mRNAs, 40 μg of total RNA was used in each sample. In assays for β-actin mRNA, 1 μg of total RNA was used for each sample.

B. In situ hybridization

Male Sprague-Dawley rats were killed by decapitation and the brains were removed and frozen in liquid isopentane (-42ºC). Subsequently, tissues were sectioned (15 µm) on a cryostat maintained at -20ºC and thawed onto polylysine-coated microscope slides. Sections were stored at -80ºC prior to tissue fixation.

Sections were removed from storage at -80ºC and placed directly on 4% buffered paraformaldehyde at room temperature. After 60 minutes, slides were washed in isotonic phosphate buffered saline (10 minutes) and treated with proteinase K (1 ug/ml in 100 mM Tris/HCl, pH 8.0) for 10 minutes at 37ºC. Subsequently, sections were subjected to successive washing in water (1 minute), 0.1 M triethanolamine (pH 8.0, plus 0.25% acetic anhydride) for 10 minutes, and 2X SSC (0.3 mM NaCl, 0.03 mM sodium citrate, pH 7.2) for 5 minutes. Sections were then dehydrated through graded alcohols and air dried.

RNA probes (as described above) were synthesized using Maxiscript RNA transcription kits (Ambion, Austin, TX). Post-fixed sections were hybridized with 1.0X10⁶ dpm of [³⁵S]UTP-labeled riboprobes in hybridization buffer containing 75% Formamide, 10% dextran sulphate, 3X SSC, 50 mM sodium phosphate buffer (pH 7.4), IX Denhardt's solution, 0.1 mg/ml yeast tRNA and 10 mM dithiothreitol in a total volume of 25 µl. The diluted probe was applied to sections on a coverslip which was sealed in place with rubber cement. The sections were hybridized overnight at 55ºC in a humid environment.

After hybridization, the rubber cement was removed and the sections were washed in 2X SSC for 5 minutes and then treated with RNase A (200 ug/ml in 10 mM Tris/HCl, pH 8.0, containing 0.5 M NaCl) for 60 minutes at 37ºC. Subsequently, the sections were washed in 2X SSC for 5 minutes, 1X SSC for 5 minutes, 0.5X SSC for 60 minutes at hybridization temperature, 0.5X SSC for 60 minutes at room temperature for 5 minutes and then dehydrated in graded alcohols and air dried. For signal detection, the sections were placed on Kodak XAR-5 X-ray film and exposed for 7 days at room temperature.

C. Results

As shown in Figure 7, based on RNase protection assays, CRF2α and CRF2ß receptor mRNAs exhibit markedly different tissue distributions. In particular, CRF2α is found primarily in the brain, particularly in the hypothalamus, lateral septum and olfactory bulb, whereas CRF2ß mRNA is found primarily, but not limited to, the heart and skeletal muscle. This is in stark contrast to the previously revealed distribution of the CRF1 receptor (Potter et al., PNAS 91:8777-8781, 1994).

Results of in situ hybridization assays are shown in Fig. 8. Fig. 8A, B, C and 8A', B', C' show coronal sections of rat brain probed with antisense CRF2α and -CRF2β probes, respectively. There is clear expression of CRF2α in the lateral septum (A), whereas CRF2β is exposed in the choroid plexus (A'). Fig. 8B and 8C show CRF2α expression in the paraventricular nucleus and the ventromedial hypothalamic nucleus, respectively. CRF2β is not detected in any of these areas (Fig. 8B' and 8C'). CRF2α is also detected in the supraoptic nuclei (Fig. 8B), whereas CRF2β is not exposed in the choroid plexus (A'). in the neighboring arterioles (Fig. 8B'). CRF2α has also been found to be expressed on many of the arterioles in the brain that express CRF2β, albeit at a lower level. (not shown). In the heart, CRF2β was the major form expressed and was found predominantly on arterioles (not shown).

EXAMPLE 6 ANATOMIC DISTRIBUTION OF CRF₁ AND CRF₂ mRNA A. Materials and Methods

1. Animals: Male Sprague-Dawley rats (200-220 g) were used for mapping studies. Prior to sacrifice, animals were maintained in a 12-hour light/dark cycle, provided with food and water ad libitum.

2. Riboprobe construction: CRF₂ cRNA riboprobe was prepared from a 460 bp cDNA fragment of the CRF₂ receptor subcloned into pBluescriptSK+ (Stratagene, La Jolla) and linearized with XbaI. The CRF₁ probe was synthesized from a 460 bp 5' fragment of CRF₁ cDNA in pBluescriptSK+ linearized with XbaI. Both CRF₁ and CRF₂ riboprobes were directed against the 5' region of their respective receptors, covering the sequence up to the third putative transmembrane region (Fig. 9). The approximate nucleotide homology between the two probes is 65% in this region. Importantly, in preliminary experiments, cRNA probes directed against the 3' region of the CRF2 receptor apparently labeled both CRF1 and CRF2 receptor mRNAs, whereas the two mRNA species could be clearly separated by 5'-specific probes under similar hybridization conditions. Thus, it appears necessary to use 5' probes for subtype-specific labeling. For both probes, specificity was confirmed by absence of signal in sections labeled with sense probes and sections pretreated with RNase prior to hybridization with antisense (cRNA) probe. CRF cRNA antisense probes were synthesized from a 779 bp fragment of CRF cDNA cloned into a pGEM3Z vector (a courtesy of Dr. Robert Thompson, University of Michigan). Riboprobes were prepared using either T3 or T7 transcription systems in a standard labeling reaction mixture consisting of: 1 µg linearized plasmid, 5X transcription buffer, 125 µCl 35S-UTP or 33P-UTP, 150 µM NTP's, 12.5 mM dithiothreitol, 20U RNAse inhibitor and 6U of the appropriate polymerase. The reaction was carried out at 37°C for 90 min. ten and the labeled probe was removed from free nucleotides via Sephadex G-50 spin columns.

3. In situ hybridization histochemistry: Dissected tissue was frozen in isopentane chilled to -42ºC and subsequently stored at -80ºC prior to sectioning on a cryostat. Tissue sections mounted on slides were then stored at -80ºC. Sections were removed from storage and placed directly in 4% buffered formaldehyde at room temperature. After 60 minutes, slides were washed in isotonic phosphate buffered saline (10 minutes) and treated with proteinase K (1 µg/ml in 100 mM Tris/HCl, pH 8.0) for 10 minutes at 37ºC. Subsequently, sections were washed in water (1 minute), 0.1 M triethanolamine (pH 8.0, plus 0.25% acetic anhydride) for 10 minutes, and 2X SSC (0.3 mM NaCl, 0.03 mM sodium citrate, pH 7.2) for 5 minutes. Sections were then dehydrated through graded alcohols and air dried. Post-fixed sections were incubated with 1.0 x 10⁶ dpm [35S]UTP-labeled riboprobes were hybridized in hybridization buffer containing 75% formamide, 10% dextran sulphate, 3X SSC, 50 mM sodium phosphate buffer (pH 7.4), 1X Denhardt's solution, 0.1 mg/ml yeast tRNA and 10 mM dithiothreitol in a total volume of 30 µl. The diluted probe was applied to sections on a glass coverslip and hybridized overnight at 55ºC in a humid environment. After hybridization, sections were washed in 2X SSC for 5 minutes and then treated with RNase A (200 µg/ml in 10 mM Tris/HCl, pH 8.0, containing 0.5 M NaCl) for 60 minutes at 37ºC. Subsequently, sections were washed in 2X SSC for 5 minutes, 1X SSC for 5 minutes, 0.1X SSC for 60 minutes at 70ºC, 0.5 X SSC at room temperature for 5 minutes, and then dehydrated in graded alcohols and air dried. For signal detection, sections were placed on Kodak Bio Max X-ray film and exposed for the required period of time or immersed in photographic emulsion (Amersham LM-1) for high resolution analysis. Autoradiograms were analyzed using automated image analysis (DAGE camera/Mac II/IMAGE program). Briefly, anatomical regions of interest were interactively selected and mean optical density measurements determined from at least 3 coronal sections. Background signal was determined from an area of the section in which no label was detectable. Immersed sections were examined under a Zeiss Axioscope.

B. Results

1. CRF2 probe selection: As illustrated in Figure 1, the CRF2 cRNA probe used in the present studies was synthesized from a 460 bp 5' fragment of CRF2 cDNA. Preliminary in situ hybridization studies using cRNA probes containing the 3' portion of the receptor (which bears high homology to the CRF1 receptor) yielded an anatomical distribution that was inconsistent with that obtained using 5'-specific riboprobes. However, the labeling pattern was consistent with both CRF1 and CRF2 receptor mRNA distribution. The high sequence homology between CRF₁ and CRF₂ receptors thus necessitates the need to use 5'-containing riboprobes for specific hybridization of CRF₂ or CRF₁ receptor subtype mRNA.

2. Anatomical distribution: The comparative anatomical distribution of CRF2 and CRF1 mRNA was determined in adjacent coronal brain sections. Table II below summarizes the semi-quantitative analysis of the data.

As illustrated in the horizontal section (Fig. 10), the distribution of CRF2 receptor mRNA differs markedly from that of CRF1 and exhibits a distinct sub-cortical pattern. While CRF1 mRNA expression was high in a range of telencephalic structures, CRF2 receptor expression exhibited a more anatomically specific pattern, including the lateral septal region (LS), bed nucleus of the stria terminalis (BNST), amygdaloid area, and olfactory bulb (Fig. 11, Table 1). The difference in expression patterns between CRF receptor subtypes was particularly evident in the septal region: CRF2 mRNA expression was very high in the lateral septal nuclei, but very low in the medial septum, the septal nucleus, where CRF1 mRNA abundance was most evident (Fig. 11B). The distribution of CRF2 receptor mRNA within the LS was, however, heterogeneous. Very high expression was evident in both the intermediate nucleus and the ventral subnuclei with only an occasional labeled cell evident in the dorsal subdivision (Fig. 12). In both the intermediate and ventral regions of the LS, the level of CRF2 receptor expression showed an obvious rostrocausal gradient, with a lower percentage of labeled cells detected in the caudal aspect of both areas. At this titer, some scattered CRF₂-expressing cells were also evident in both the vertical and also horizontal extremities of the diagonal band. Again, in contrast to the higher titers of CRF₁ mRNA found in these regions (Fig. 11). Differential patterns of CRF subtype expression were also evident within the olfactory bulb (Fig. 11A). Here, CRF₁ receptor expression was particularly high in the inner granular cell and mitral cell layer with lower titers detectable in the external granular layer and ependyma. However, most cells expressing CRF₂ receptors were found in the ventricle within the ependymal area and inner granular cell layer (Fig. 11A). Both CRF₁ and CRF₂ receptors were also found in the accessory olfactory nucleus.

Both CRF2 and CRF1 receptor mRNA expression was evident in the BNST, particularly in the medial aspect and the amygdale area. Within the BNST, CRF2 receptor expression appeared lower in the lateral regions, where the greatest abundance of CRF-expressing cells was found (Fig. 13A and B). CRF2 receptor expression was found in both medial and lateral units of the BNST (Fig. 13C). Within the amygdale, the highest titers of CRF2 receptor expression were evident in the cortical and medial amygdale nuclei, with lower expression in the basolateral nucleus (Fig. 11C). Complementarily, CRF1 receptor expression was very high in the basolateral area but low in the cortical amygdala (Fig. 14). Both CRF1 and CRF2 receptor mRNA expression was not evident in the central nucleus. Within the hippocampal formation, CRF2 receptors were generally expressed at low to moderate titers in pyramidal cells of CA subfields and granule cells of the dentate gyrus. Scattered cells with high titers of CRF2 expression were evident in non-granule cell layers in the ventral dentate gyrus (Fig. 15). CRF1 receptor expression was most pronounced in the pyramidal cell layer of CA3/4 with moderate titers evident in CA1. CRF1 receptor expression was apparently absent in the dorsal dentate gyrus (Fig. 15). Interestingly, high titers of CRF1 expression were evident in the ventral hippocampus compared with the dorsal aspect (Fig. 11C and D). Cells expressing both CRF1 and CRF2 were found throughout the entorhinal cortex.

The distribution of CRF2 receptor transcripts in diencephalic structures was mainly restricted to the hypothalamus, where labelled cells were evident in preoptic, anterior and tuberal regions. The level of CRF2 mRNA expression expressed in the venous The CRF2 receptor expression found in the dorsomedial hypothalamic nuclei (VMH) was among the highest found in the brain (Fig. 16). Both dorsal and ventral aspects of the VMH showed high titers of CRF2 mRNA relative to other brain regions, although CRF2 mRNA expression was most evident in the dorsomedial area. At this level, however, CRF1 receptor expression was predominantly localized in the dorsomedial hypothalamic nucleus (DMH), with only a limited number of labeled cells in the VMH. In the anterior hypothalamus, CRF2 mRNA was localized to highly expressing cells in the supraoptic nucleus (SO) and medial areas of the paraventricular nucleus (PVN) (Fig. 17, Fig. 18). Within the PVN, CRF2 mRNA appeared to be expressed in medial parvocellular cells, partially coincident with CRF mRNA expression (Fig. 10A). This raises the interesting possibility that CRF2 receptors in this nucleus may act as autoreceptors in selected cells. In both the SO and the PVN, the number of cells expressing CRF1 mRNA was extremely low (Fig. 17, Fig. 18). Cells expressing CRF2 mRNA were also evident in the anterior and lateral hypothalamic areas, the suprachiasmatic nucleus, and the medial preoptic area (MPA).

In the midbrain, CRF2-expressing cells were evident within nuclei biochemically characterized as serotonin-containing nuclei. Strongly hybridizing cells were located along the midline in the dorsal raphe nucleus (DR), the caudal linear nucleus, and in the lateral aspects of the central gray (Fig. 19). More ventrally, CRF2 receptor expression was also detectable in the medial raphe (MR) and the interpedunuclear nucleus (IPN), particularly within the rostral subnucleus (Fig. 19). With the exception of the IPN, CRF1 receptor mRNA titers were generally low in all of these areas. At this level, CRF2-expressing cells were also evident in clusters in the lateral and dorsal regions of the inner colliculus. However, higher titers of CRF1 mRNA expression were also present in the inferior colliculus. Higher titers of CRF₁ mRNA were also found in the visual receptive fields of the superior colliculus, where CRF₂ receptor expression was very low. This differential expression pattern was repeated in other sensory relay structures of the brainstem. Thus, CRF₂ receptor expression was restricted to a few scattered cells in these structures (Table 2), whereas the level of CRF₁ receptor mRNA abundance was high in tegmental nuclei and sensory trigeminal areas. Similarly, in the cerebellum, CRF₁ receptor mRNA titers were very high in Purkinje and granule cell layers, where whereas CRF2 receptor mRNA was present in granule cells at very limited titres (Fig. 11E).

In non-neuronal structures, very high levels of CRF2 receptor expression were evident in the choroid plexus of the third, fourth, and lateral ventricles (Fig. 20). In addition, cerebral arterioles consistently expressed CRF2 mRNA in all brain levels examined (Fig. 21). CRF1 mRNA was detectable near background levels in these structures. In the pituitary, CRF1 expression was detectable in both the anterior and intermediate lobes with particularly high expression in clusters within the anterior lobe (Fig. 22). In the anterior lobe, CRF2 receptor expression was only detectable in scattered cells (Fig. 22).

C. Discussion

The present in situ hybridization studies indicate that at least two CRF receptor subtypes, CRF1 and CRF2, are expressed in the mammalian rat brain. The heterogeneous anatomical distribution patterns of CRF1 and CRF2 mRNA expression suggest distinct functional roles for each receptor in CRF-related CNS circuits. While CRF1 receptor expression was most abundant in neocortical, cerebellar, and sensory relay structures, CRF2 receptor expression was generally localized to specific subcortical structures, including the lateral septum and various hypothalamic nuclei.

In the forebrain, the highest titers of CRF2 receptor expression were found in lateral septal nuclei. The lateral septum, thanks to widely dispersed reciprocal connections within the brain, is involved in a variety of physiological processes ranging from higher cognitive functions such as learning and memory to autonomic regulation including food and water intake. In addition, the septum plays a central role in classical limbic circuits and is thus important in a variety of emotional states, including fear and aggression. The lateral septal nuclei receive CRF-containing afferents from rostral hypothalamic regions, particularly the anterior hypothalamic area (Sakanaka et al., j. Comp. Neurol. 270:404-415, 1988). Interestingly, the majority of these CRF-like immunoreactive fibers are found in the most lateral aspects of the LSV and the LSI (Sakanaka et al., J. Comp. Neurol. 270:404-415, 1988), with septal subnuclei containing the highest titers of CRF₂ receptor mRNA (Fig. 12). The lack of CRF₁ receptor expression in these nuclei suggests that CRF₂ receptors alone mediate the postsynaptic effects of CRF inputs to this region. The major GABAergic neurons of the lateral septum provide inhibitory input to hypothalamic regions as well as amygdala nuclei (Jakab et al., 1991, Calbindin-containing somatospiny neurons of the rat lateral septal area project to the medial amygdala and the anterior hypothalamus, Third IBRO World Congress Neuroscience Abstracts, 324) and receive excitatory glutamate input from the hippocampal formation (Joels and Urban, Experimental Brain Research 54:455-462, 1984). The lateral septum thus acts both as an integrator of limbic circuits and as an interface between telencephalic and diencephalic areas. The high level of CRF2 receptor expression in this area indicates that CRF2 receptors modulate limbic circuits at the level of septal activity.

In agreement with previous in situ hybridization studies (Potter et al., Proceedings of the National Academy of Sciences 91:8777-8781, 1994), a general lack of CRF1 receptor expression was found in hypothalamic nuclei. From the present studies, it is clear that CRF2 receptor mRNA was evident within the rostrocaudal perimeter of the hypothalamus, whereas CRF1 receptor expression was restricted. The difference in the level of CRF receptor subtype expression was particularly evident in the paraventricular nucleus, where CRF2 receptor expression was readily detectable, whereas CRF1 receptor mRNA was present only in scattered cells. The distribution of cells expressing CRF2 receptor mRNA within the PVN is consistent with the cellular distribution of CRF mRNA (Fig. 18), indicating an autoreceptor role for CRF2 receptors in this nucleus. The CRF neurons of the PVN play a classical hypophysiotropic role in controlling ACTH release from the anterior pituitary (Wiegand and Price, J. Comp. Neurol. 192:1-19, 1980) and as such are central to the control of the hypothalamo-pituitary-adrenal system in mammals. In addition to this stress axis-related role, subpopulations of PVN CRF neurons, particularly within the dorsal parvocellular region and ventral aspect of the medial parvocellular region (mpv), project to autonomous cell groups in the brain stem and spinal cord (Sawchenko, Brain Res. 437, 1987). Thus, the high level of CRF2 receptor expression suggests a presynaptic role for CRF2 receptors in modulating autonomic-related CRF projection neurons.

Selective expression of CRF2 receptor mRNA was also evident in the magnocellular neurosecretory neurons of the supraoptic nucleus. In view of the putative connection of CRF2 receptor expression with CRF neurons in the PVN, it is notable that a subset of SO neurons also synthesize CRF (Kawata et al., Cell Tissue Research 230:239-246, 1983). The presence of CRF2 receptors in both SO and PVN neurons indicates that these sites may act to influence hypothalamic input to both the anterior and posterior pituitary. Within the pituitary, however, CRF1 receptor expression predominates over CRF2 expression in both the intermediate and anterior lobes. CRF2 receptors may thus, in terms of HPA axis activity, mediate CRF effects at the level of the hypothalamus, whereas CRF1 receptors are responsible for CRF-induced changes in ACTH release in pituitary corticotropes.

Within the caudal hypothalamus, CRF1 and CRF2 receptors exhibit mutually exclusive patterns of mRNA distribution: CRF1 receptor mRNA is abundant in the dorsomedial nucleus but low within the VMH, whereas CRF2 receptor mRNA expression was very high within the VMH but undetectable within the DMH. CRF-containing fibers originating from the corticomedial amygdala and subiculum terminate within the VMH (Sakanaka et al., Brain Res. 382:213-238, 1986). Afferents from the VMH in turn innervate the septum, BNST and amygdala, as well as brainstem regions (Simerly, RB, Anatomical substrates of hypothalamic integration. In: The Rat Nervous System (Paxinos, G., ed.), pp. 353-376, Academic Press, 1995). Microinjection of CRF into the VMH is associated with changes in both autonomic outflow and gastrointestinal function (Brown and Fisher, Regulation of the autonomic nervous system by corticotropin-releasing factor. In: Corticotropin-releasing factor: Basic and clinical studies of a neuropeptide (De Souza, EB, Nemeroff, CB, eds.), pp. 291-298, Boca Raton, FL, CRC Press, Inc., 1990; Tache et al., CRF: Central nervous system action to influence gastrointestinal function and role in the gastrointestinal response to stress. In: Corticotropin-releasing factor: Basic and clinical studies of a neuropeptide (De Souza, EB, Nemeroff, CB, eds.), pp. 299-308, Boca Raton, FL, CRC Press, Inc., 1990). The high level of CRF₂ receptor expression in the VMH implies that CRF receptor subtype as a terminal or somato-dendritic regulator of these CRF-related physiological functions. Furthermore, dysregulation of CRF2 receptors in this locus, or the PVN, may participate in the proposed role of central CRF systems in the development of obesity/appetite-reducing syndromes (Krahn and Gosnell, Psychiatric Medicine 7:235-245, 1989).

In addition to CRF involvement in the development of eating disorders, there is a high level of evidence that these neuropeptides are involved in the pathophysiology of affective disorders such as anxiety and depression. For example, CRF injected into the locus coeruleus of rodents produces an anxiogenic response, while benzodiazepine anxiolytics have been shown to reduce CRF concentration in the same nucleus (Owens et al., J. Pharmacol. Exp. Ther. 258:349-356, 1991). In clinical studies of major depression, patients have been found to exhibit signs of CRF hypersecretion, including: increased CRF concentrations in CSF, increased HPA activity, a blunted ACTH response to CRF, and pituitary and adrenal hypertrophy (Owens and Nemeroff, The role of corticotropin-releasing factor in the pathophysiology of affective and anxiety disorders: laboratory and clinical studies. In: Corticotropin-Releasing Factor (Chadwick, D.J. Marsh, J., Ackrill, K., eds.) pp. 296-316, John Wiley and Sons, 1993). Given the stimulatory role of CRF in HPA activity, it remains possible that the hypercortisolemia observed in depression results from increased central CRF drive. The possibility that hyperactivity of brain CRF circuits contributes to the symptomatology of depressive illness is supported by preclinical studies in both rodents and non-human primates. In both species, central administration of CRF produces a spectrum of behavioral responses reminiscent of depressive illness in humans (Koob and Britton, Behavioral effects of corticotropin-releasing factor. In: Corticotropin-releasing factor: Basic and clinical studies neuropeptide (De Souza, E.B., Nemeroff, C.B., eds.), Boca Raton, FL, CRC Press, Inc., 1990; Kalin, Behavioral and endocrine studies of corticotropin-releasing hormone in primates. In: Corticotropin-releasing factor: Basic and clinical studies of a neuropeptide (De Souza, E.B., Nemeroff, C.B., eds.), pp. 275-290, Boca Raton, FL, CRC Press, Inc., 1990). While the specific underlying mechanisms by which CRF elicits such behavioral responses remain largely undefined, it is likely that modulation of limbic circuits in the brain and engagement of specific populations of CRF neurons are involved (Rainnie et al., J. Pharm. Exp. Therap. 263: 846-858, 1992).

In this regard, the present study provides an anatomical basis for the involvement of both CRF1 and CRF2 receptors in mediating limbic CRF actions. While the CRF2 receptor can be considered the predominant hypothalamic CRF receptor, CRF1 and CRF2 receptors have been localized in classical limbic structures, such as the amygdaloid complex, hippocampus, and septal nuclei.

In addition to neuroendocrine abnormalities, a compelling body of data indicates dysfunction in central serotonergic activity in depressive illness. From postmortem studies it is clear that serotonin metabolism and specific receptor subtypes are altered in some brain regions in depressed patients (Meltzer, Br. J. Psychiat. 155:25-31, 1989; Yates et al., Psychiatry 27:489-496, 1990). Drugs that inhibit serotonin metabolism, inhibit serotonin uptake from the synapse, or act to directly stimulate serotonin receptors are all effective antidepressants (Peroutka and Snyder, Science 210:88-90, 1980; Traber and Glaser, Trends Pharmacol. Sci 8:432-437, 1987). Thus, since both the HPA axis and the serotonergic system play a role in affective illness, it is likely that interactions between these two systems may be relevant to the pathophysiology and pharmacotherapy of depression (Chalmers et al., Clin. Neurosci. 1:122-128, 1993). In this context, the present data indicate a selective expression of CRF2 receptor mRNA in serotonergic cell body nuclei in the midbrain. Both dorsal and median raphenuclei express CRF2 mRNA, as do cells in serotonin-associated regions of the interpeduncular nucleus and central gray matter (Fig. 19). Since the raphenuclei receive CRFergic input from forebrain regions (Sawchenko and Swanson, Organization of CRF immunoreactive cells and fibers in the rat brain: immunohistochemical studies. In: Corticotropin-releasing factor: Basic and clinical studies of a neuropeptide (De Souza, E.B., Nemeroff, C.B., eds.), pp. 29-52, Boca Raton, FL, CRC Press Inc., 1990), it is likely that any CRF-induced modulation of serotonergic activity is mediated by CRF2 receptor. Such an interaction provides an anatomical and biochemical basis for the central "stress"-related modulation of serotonergic function and a foundation for theories of stress-induced affective disorders.

In addition to neuronal localization, the present study also indicates high levels of CRF2 receptor expression in both the choroid plexus and cerebral arterioles. The presence of signal in both structures may indicate endothelial cell localization. The CRF2 cRNA probe used in the present study does not differentiate between two obvious splice forms of the CRF2 receptor, CRF2α and CRF2β (Lovenberg et al., Proc. Natl. Acad Sci USA 92:836-840, 1995). However, preliminary data indicate that the CRF2β form of the receptor may predominate in blood vessels. Interestingly, CRF2β is also expressed in peripheral tissues such as heart, lung, and skeletal muscle, where it may function to mediate vascular effects of CRF.

In summary, a differential cellular distribution of CRF2 and CRF1 receptor RNA has been identified in the rat brain and pituitary. This distribution suggests that the CRF1 receptor is the primary neuroendocrine pituitary CRF receptor and plays a predominant role in the cortical, cerebellar, and sensory roles of CRF in the brain. The regional anatomical distribution of CRF2 receptor mRNA suggests a role for this receptor in hypothalamic endocrine, autonomic, and behavioral actions of brain CRF. The presence of CRF2 receptor mRNA in the hypothalamic PVN and medial and cortical amygdaloid regions may indicate an autoreceptor role for this site in selected circuits. Table II Semiquantitative assessment of CRF₁ and CRF₂ receptor mRNA distribution in rat brain and pituitary

The amount of CRF1 and CRF2 mRNA was determined for each anatomical region from optical density measurements. Density values for each parameter are presented according to their respective percentage distributions: ++++ (> 75%), very dense; +++ (< 75%, > 50%), dense; ++ (< 50%, > 25%), moderate; + (25%, > 10%), low; -/+ (< 10%), scattered cells.

SEQUENCE LOG

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(i) APPLICANT: Lovenberg, Timothy W.

Oltersdorf, Tilman

Liaw, Chen

Grigoriadis, Dimitri E.

Chalmers, Derek T.

DeSouza, Errol B.

(ii) TITLE OF THE INVENTION: CORTICOTROPIN RELEASE FACTOR 2 RECEPTORS

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Claims (43)

1. An isolated nucleic acid molecule encoding a CRF₂ receptor, wherein said CRF₂ receptor is encoded by: (a) a nucleic acid sequence comprising the coding region of any of the sequences with Sequence ID Nos. 1, 3 or 7 or a derivative thereof with more than 70% homology with said coding region; (b) a nucleic acid sequence capable of hybridising under conditions of high stringency to a nucleic acid sequence complementary to (a); or (c) nucleic acid sequences which are degenerate as a result of the genetic code for CRF2 receptors encoded by a nucleic acid sequence defined in (a) or (b). 2. The isolated nucleic acid molecule of claim 1, which comprises the nucleotide sequence in Sequence I.D. No. 3, from nucleotide No. 216 to nucleotide No. 1,448. 3. The isolated nucleic acid molecule of claim 1, wherein said molecule encodes a protein having the amino acid sequence of Sequence I.D. No. 4, from amino acid No. 1 to amino acid No. 411. 4. The isolated nucleic acid molecule of claim 1, wherein said molecule encodes a rat CRF2 receptor. 5. The isolated nucleic acid molecule of claim 1, wherein said molecule encodes a human CRF₂ receptor. 6. An isolated nucleic acid molecule encoding an N-terminal CRF₂ extracellular domain, wherein said N-terminal CRF₂ extracellular domain is encoded by: (a) a nucleic acid sequence comprising the coding region of the N-terminal extracellular domain defined by nucleotide No. 44 to nucleotide No. 457 of Sequence I.D. No. 1, nucleotide No. 216 to nucleotide No. 569 of Sequence I.D. No. 3 or the equivalent region of Sequence I.D. No. 7, or a derivative thereof having more than 70% homology with said coding region; (b) a nucleic acid sequence capable of hybridising under conditions of high stringency to a nucleic acid sequence complementary to (a); or (c) nucleic acid sequences which are degenerate as a result of the genetic code for CRF2 receptors encoded by a nucleic acid sequence defined in (a) or (b). 7. The isolated nucleic acid molecule of claim 6, which comprises the nucleotide sequence in Sequence I.D. No. 3, from nucleotide No. 216 to nucleotide No. 569. 8. The isolated nucleic acid molecule of claim 6, wherein said molecule encodes a protein having the amino acid sequence of Sequence I.D. No. 4, from amino acid No. 1 to amino acid No. 118. 9. A recombinant expression vector comprising a promoter operably linked to a nucleic acid molecule according to any one of claims 1 to 9. 10. A recombinant viral vector capable of directing the expression of a nucleic acid molecule according to any one of claims 1 to 9, wherein said viral vector is selected from the group consisting of retroviral vectors, adenoviral vectors and herpes simplex virus vectors. 11. A host cell containing a recombinant expression vector according to claim 9 or 10. 12. An isolated CRF2 receptor or variant thereof encoded by a nucleic acid sequence according to claim 1. 13. The isolated CRF2 receptor of claim 12, having the amino acid sequence of Sequence I.D. No. 4, from amino acid No. 1 to amino acid No. 411. 14. An isolated N-terminal CRF2 receptor extracellular domain or variant thereof, encoded by a nucleic acid sequence according to claim 6. 15. The isolated CRF2 receptor of claim 14, having the amino acid sequence of Sequence I.D. No. 4, from amino acid No. 1 to amino acid No. 118. 16. An isolated antibody capable of specifically binding to a CRF2 receptor or variant thereof encoded by a nucleic acid sequence according to claim 1. 17. An isolated antibody capable of specifically binding to an N-terminal extracellular CRF2 receptor domain encoded by a nucleic acid sequence according to claim 6. 18. The antibody of claim 16 or 17, wherein said antibody is selected from the group consisting of monoclonal antibodies and antibody fragments. 19. The antibody of claim 16 or 17, wherein said antibody is a polyclonal antibody. 20. The antibody of claim 16 or 17, wherein said antibody is capable of blocking the binding of CRF to a CRF2 receptor. 21. The antibody of claim 16 or 17, wherein said antibody is a murine antibody. 22. The antibody of claim 16 or 17, wherein said antibody is a human antibody. 23. A hybridoma producing an antibody according to any one of claims 16 to 18 and 20 to 22. 24. A nucleic acid probe of at least 18 nucleotides in length capable of hybridizing specifically to (a) a nucleic acid sequence encoding a CRF2 receptor according to claim 1, or (b) a nucleic acid sequence complementary to a nucleic acid sequence according to claim 1, under conditions of moderate or high stringency, but not to CRF receptor nucleic acids whose sequences have been submitted to the Gen Bank database (accession numbers L23332, L23333 and L24096, respectively) and to the EMBL Data Library (accession numbers X72305 and X72304, respectively). 25. The nucleic acid probe of claim 24, further comprising a label. 26. The nucleic acid probe of claim 25, wherein said label is selected from the group consisting of radioactive labels, fluorescent labels, enzymatic labels and chromogenic labels. 27. A method for detecting the presence of a compound that binds to a CRF₂ receptor, which comprises: (a) exposing one or more compounds to cells or cell membranes expressing CRF₂ receptors encoded by a nucleic acid sequence according to claim 1 under conditions and for a time sufficient to allow binding of said compounds to said receptors; and (b) isolating compounds which bind to said receptors so that the presence of a compound which binds to a CRF₂ receptor can be detected. 28. A method for detecting the presence of a compound that binds to a CRF₂ receptor, which comprises: (a) exposing one or more compounds to an N-terminal extracellular CRF₂ receptor domain encoded by a nucleic acid sequence according to claim 6 under conditions and for a period of time sufficient to allow binding of a compound to the N-terminal extracellular domain; and (b) isolating compounds that bind to said N-terminal CRF2 receptor extracellular domain so that the presence of a compound that binds to a CRF2 receptor can be detected. 29. The method of claim 28, wherein said compounds are labeled with an agent selected from the group consisting of fluorescent molecules, enzymes and radionuclides. 30. A method for determining whether a selected compound is a CRF₂ receptor agonist or antagonist, which comprises: (a) exposing a selected compound to cells expressing CRF2 receptors encoded by a nucleic acid sequence according to claim 1 under conditions and for a period of time sufficient to allow binding of the compound and an associated response through a pathway; and (b) detecting either an increase or decrease in the activity of the pathway compared to the activity of the pathway prior to step (a), and thereby determining whether said selected compound is a CRF₂ receptor agonist or antagonist. 31. A method according to claim 30, further comprising the step of isolating compounds that either increase or decrease the activity of the pathway so that the presence of a CRF₂ receptor agonist or antagonist can be detected. 32. The method of claim 30, wherein the detecting step comprises detecting a reduction in the activity of the pathway resulting from binding of the compound to the CRF2 receptor relative to stimulation of the pathway by the CRF2 receptor agonist alone, and determining therefrom the presence of a CRF2 antagonist. 33. The method of claim 30, wherein the detecting step comprises detecting an increase in the activity of the pathway resulting from binding of the compound to the CRF2 receptor and determining therefrom the presence of a CRF2 receptor agonist. 34. The method of any one of claims 30 to 33, wherein said pathway is the adenylate cyclase pathway. 35. A CRF2 receptor antagonist for use in therapy, wherein said CRF2 receptor is encoded by a nucleic acid sequence according to claim 1. 36. A CRF2 receptor agonist for use in therapy, wherein said CRF2 receptor is encoded by a nucleic acid sequence according to claim 1. 37. Use of a CRF2 receptor antagonist for the manufacture of a medicament for the treatment of cerebrovascular diseases, wherein said CRF2 receptor is encoded by a nucleic acid sequence according to claim 1. 38. Use of a CRF2 receptor antagonist according to claim 37, wherein said cerebrovascular disease is selected from the group consisting of stroke, reperfusion injury and migraines. 39. Use of a CRF₂ receptor antagonist for the manufacture of a medicament for the treatment of learning or memory disorders, wherein said CRF₂ receptor is encoded by a nucleic acid sequence according to claim 1. 40. Use of a CRF2 receptor antagonist for the manufacture of a medicament for the treatment of Alzheimer's disease, wherein said CRF2 receptor is encoded by a nucleic acid sequence according to claim 1. 41. Use of the CRF2 receptor antagonist according to any one of claims 37 to 40, wherein said antagonist is α-helical oCRF(9-41) or d-Phe-r/h-CRF(12-41). 42. Use of a CRF₂ receptor for the manufacture of a medicament for modulating metabolism and/or satiety, wherein said CRF₂ receptor is encoded by a nucleic acid sequence according to claim 1. 43. Use of a CRF2 receptor antagonist for the manufacture of a medicament for the treatment of anorexia nervosa, wherein said CRF2 receptor is encoded by a nucleic acid sequence according to claim 1.